Methods of treatment for guillain-barre syndrome

ABSTRACT

This invention relates generally to methods of treatment for Guillain-Barre&#39; Syndrome (GBS) and, more specifically, to methods involving the inhibition of the classical pathway of complement activation.

CROSS REFERENCES TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/823,876, filed May 15, 2013, which is hereby incorporated byreference in its entirety.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name: 717192000740SeqList.txt,date recorded: May 14, 2014, size: 23 KB).

BACKGROUND

1. Field

This invention relates generally to methods of treatment forGuillain-Barré Syndrome and more specifically to methods involving theinhibition of the classical pathway of complement activation.

2. Description of Related Art

Guillain-Barre' syndrome (GBS) represents a spectrum of acuteidiopathic, usually monophasic peripheral neuropathies (Willison andYuki, 2002; Hughes and Cornblath, 2005; van Doom et al., 2008). GBStypically presents as an ascending paralysis with weakness beginning inthe feet and hands and migrating towards the trunk. As the weaknessprogresses upward, usually over periods of hours to days, the arms andfacial muscles can also become affected. Frequently, the lower cranialnerves may be affected, leading to bulbar weakness, oropharyngealdysphagia (drooling, or difficulty swallowing, and/or maintaining anopen airway) and life-threatening respiratory difficulties. There is amortality rate of 2-3% with most patients requiring hospitalization, andabout 30% needing ventillatory assistance for treatment of respiratoryfailure (Burt et al. 2009). Nearly 80% of patients have a completerecovery within a few months to a year, although minor findings maypersist, such as areflexia. About 5-10% of patients have one or morelate relapses, in which case they are then classified as having chronicinflammatory demyelinating polyneuropathy.

Current pharmacologic therapy consists of either plasmapheresis (i.e.,filtering antibodies out of the blood stream) or administration ofintravenous immunoglobulins (IVIg). These two treatments are equallyeffective; however, a combination of the two is not significantly betterthan either alone. Plasmapheresis hastens recovery when used within fourweeks of the onset of symptoms (Hughes et al 2003). IVIg has equivalentefficacy to plasmapheresis when started within two weeks of the onset ofsymptoms, and has fewer complications (Hughes et al. 2003). IVIg isusually used first because of its ease of administration and safetyprofile. However, the use of IVIg is not without risk; occasionally itcauses hepatitis, or in rare cases, renal failure if used for longerthan five days, and can also cause clotting abnormalities. Despite theavailability of these therapies and improvements in supportive care,20-30% of patients are left with some form of permanent disability,5-10% of patients are left with severe disability (such as inability towalk unassisted), and 2-3% of patients die. Accordingly, there is acontinuing need for new therapies to treat GBS.

Advances in the understanding of the immunopathogenesis of the diseasehave identified new targets for therapeutic intervention including thecomplement pathway, which is a fundamental component of the innateimmune system. In brief, an aberrant immune response to myelin and/oraxolemmal antigens, typically following an infection, is considered tobe the fundamental cause of GBS (van Doorn et al. 2008). Human andanimal model studies suggest that pathogen infection results in theproduction of antibodies to the pathogen gangliosides, which cross-reactwith host gangliosides that are abundant in neuronal cell membranes.Accumulation of such antibodies at the pre-synaptic membrane of motorneurons results in activation of the complement cascade, the formationof complement membrane attack complex (MAC), and recruitment ofmacrophages. The ensuing ultrastructural destruction and blockade ofsynaptic transmission at the neuromuscular junction can causes themuscle weakness associated with the disease (Fewou et al. 2014).

Evidence for the important role of the complement pathway in GBS comesfrom a variety of patient tissue studies and from animal models of GBSsubtypes like Miller Fisher Syndrome (MFS). Studies of spinal roots andnerves from autopsy cases of the GBS subtype acute motor axonalneuropathy (AMAN) revealed the presence of infiltrating macrophages withextensive processes in the periaxonal space abutting the nodal andinternodal axolemma and displacing the adaxonal Schwann cell membraneand myelin sheath. Approximately 50% of AMAN patients (Ho et al. 1999)and 80-90% of MFS patients (Willison and Yuki 2002) are positive foranti-ganglioside antibodies and immunocytochemistry analysis showed veryintense immunoglobulin G (IgG) and complement C3d and C5b-9 (membraneattack complex [MAC]) deposits bound to the nodal and internodalaxolemma in the periaxonal space. Additional studies have also shown theactivation of complement products in plasma and CSF of GBS patients(Sanders et al., 1986; Hartung et al., 1987; Koski et al., 1987;Hafer-Macko et al., 1996). Yuki et al. recapitulated some of the keypathological findings in rabbits sensitized with gangliosides includingGM1 (Yuki et al., 2001). In these animals, high anti-GM1 IgG antibodytitres were observed, accompanied by a flaccid limb weakness. Peripheralnerves showed predominant Wallerian-like degeneration with neitherlymphocytic infiltration nor demyelination, whereas IgG was deposited onventral root axons. Macrophage infiltration into the periaxonal spaceand nodal complement deposits have also been demonstrated (Susuki etal., 2003). Recently, some of the molecular events that may lead toaxonal conduction block have been defined, which include thedisappearance of sodium channel immunoreactivity at the node of Ranvier(Susuki et al., 2007). It has also recently been shown that inhibitionof MAC assembly using a monoclonal antibody inhibitor of C5 in a mousemodel of MFS had a major neuroprotective effect, completely preventingany structural and functional changes at the node of Ranvier, foundpreviously at the motor nerve terminal (Halstead et al., 2008).

Recently, clinical trials have begun in GBS for Eculizumab, which is aterminal complement inhibitor currently approved for the treatment ofparoxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic uremicsyndrome (aHUS) (see e.g., FDA drug label for Eculizumab). WhileEculizumab was shown to provide clinical benefits in animal models, oneproblem with inhibiting the terminal complement pathway is that suchinhibition can cause serious side-effects due to the associatedweakening of a patient's immune defenses against bacterial infections.Eculizumab therapy, for example, is associated with elevated risks ofmeningococcal meningitis and other bacterial infections (see, e.g. FDAdrug label for Eculizumab). Moreover, Eculizumab is not expected toprevent complement-dependent cell-mediated cytotoxicity (CDCC) to theextent that CDCC is driven by complement factor C3a, an anaphalotoxinproduced upstream of the Eculizumab targeted C5-convertase (Klos et al.2009).

The C1 complex is the initiating factor of the classical complementcascade, and binds directly to autoantibody complexes. C1q binding toantibody leads to the activation of the C1r and C1s enzymes leading tothe production of anaphylatoxins (C3a, C4a, and C5a), and the membraneattack complex (MAC). In contrast to Eculizumab, which blocks C5a andMAC formation for all three arms of the complement system, inhibition ofC1 may specifically block C5a and MAC formation that is dependentprimarily upon classical pathway activators (e.g., antibodies), whichwould leave the lectin and alternate pathways active for fightinginfection. In addition to this safety advantage for C1 inhibition, C1blockade may be more effective inhibitor of harmful autoantibodyresponses because it can block MAC formation as well as the productionof opsins (i.e., C3b) and inflammatory mediators (i.e., C3a, C4a) thatmay contribute to the pathogenesis of the disease, and which are notblocked by Eculizumab.

Accordingly, the C1 complex may be an attractive therapeutic target forGBS. Thus, there is a need to develop new treatment options for GBSpatients, such as antibodies that inhibit the C1 complex and itscomponents, which would then inhibit the early stages of complementactivation, including the classical complement activation pathway.

All references cited herein, including patent applications andpublications, are hereby incorporated by reference in their entirety.

BRIEF SUMMARY

Certain aspects of the present disclosure provide anti-C1q, anti-C1s,anti-C1r, and anti-C1 complex antibodies and methods of using suchantibodies for creating or preventing Guillain-Barré Syndrome (GBS) inan individual.

Certain aspects of the present disclosure are directed to methods oftreating Guillain-Barre' syndrome (GBS) that include inhibiting theclassical pathway of complement activation by neutralizing thecomplement factors C1q, C1r, or C1s, e.g., through the administration ofantibodies, such as monoclonal, chimeric, humanized antibodies, antibodyfragments, etc., which bind to one or more of these complement factors.

In certain aspects, the present disclosure provides a method of treatingor preventing Guillain-Barré Syndrome (GBS) in an individual, comprisingadministering to the individual a therapeutically effective amount of anantibody, wherein the antibody is: i) an anti-C1q antibody, wherein theanti-C1q antibody inhibits the interaction between C1q and anautoantibody, or between C1q and C1r, or between C1q and C1s, or whereinthe anti-C1q antibody prevents C1q from activating C1r or C1s; ii) ananti-C1r antibody, wherein the anti-C1r antibody inhibits theinteraction between C1r and C1q, or between C1r and C1s, or wherein theanti-C1r antibody inhibits the catalytic activity of C1r or inhibits theprocessing of pro-C1r to an active protease; iii) an anti-C1s antibody,wherein the anti-C1s antibody inhibits the interaction between C1s andC1q, or between C1s and C1r, or between C1s and C2, or between C1s andC4, or wherein the anti-C1s antibody inhibits the catalytic activity ofC1s or inhibits the processing of pro-C1s to an active protease; or iv)an anti-C1 complex antibody that binds to a combinatorial epitope withinthe C1 complex, wherein said combinatorial epitope is comprised of C1qand C1s; C1q and C1r; C1r and C1s; or C1q, C1r, and C1s; or wherein theanti-C1 complex antibody inhibits C1r or C1s activation or preventstheir ability to act on C2 or C4. In other aspects, the presentdisclosure provides an antibody for use in treating or preventingGuillain-Barré Syndrome (GBS) in an individual, wherein the antibody is:i) an anti-C1q antibody, wherein the anti-C1q antibody inhibits theinteraction between C1q and an autoantibody, or between C1q and C1r, orbetween C1q and C1s, or wherein the anti-C1q antibody prevents C1q fromactivating C1r or C1s; ii) an anti-C1r antibody, wherein the anti-C1rantibody inhibits the interaction between C1r and C1q, or between C1rand C1s, or wherein the anti-C1r antibody inhibits the catalyticactivity of C1r or inhibits the processing of pro-C1r to an activeprotease; iii) an anti-C1s antibody, wherein the anti-C1s antibodyinhibits the interaction between C1s and C1q, or between C1s and C1r, orbetween C1s and C2, or between C1s and C4, or wherein the anti-C1santibody inhibits the catalytic activity of C1s or inhibits theprocessing of pro-C1s to an active protease; or iv) an anti-C1 complexantibody that binds to a combinatorial epitope within the C1 complex,wherein said combinatorial epitope is comprised of C1q and C1s; C1q andC1r; C1r and C1s; or C1q, C1r, and C1s; or wherein the anti-C1 complexantibody inhibits C1r or C1s activation or prevents their ability to acton C2 or C4. In other aspects, the present disclosure provides use of anantibody in the manufacture of a medicament for treating or preventingGuillain-Barré Syndrome (GBS) in an individual, wherein the antibody is:i) an anti-C1q antibody, wherein the anti-C1q antibody inhibits theinteraction between C1q and an autoantibody, or between C1q and C1r, orbetween C1q and C1s, or wherein the anti-C1q antibody prevents C1q fromactivating C1r or C1s; ii) an anti-C1r antibody, wherein the anti-C1rantibody inhibits the interaction between C1r and C1q, or between C1rand C1s, or wherein the anti-C1r antibody inhibits the catalyticactivity of C1r or inhibits the processing of pro-C1r to an activeprotease; iii) an anti-C1s antibody, wherein the anti-C1s antibodyinhibits the interaction between C1s and C1q, or between C1s and C1r, orbetween C1s and C2, or between C1s and C4, or wherein the anti-C1santibody inhibits the catalytic activity of C1s or inhibits theprocessing of pro-C1s to an active protease; or iv) an anti-C1 complexantibody that binds to a combinatorial epitope within the C1 complex,wherein said combinatorial epitope is comprised of C1q and C1s; C1q andC1r; C1r and C1s; or C1q, C1r, and C1s; or wherein the anti-C1 complexantibody inhibits C1r or C1s activation or prevents their ability to acton C2 or C4.

In other aspects, the present disclosure provides a method of treatingor preventing Guillain-Barré Syndrome (GBS) in an individual, comprisingadministering to the individual a therapeutically effective amount of ananti-C1q antibody wherein the antibody is: i) an isolated anti-C1qantibody comprising a light chain variable domain and a heavy chainvariable domain, wherein the light chain variable domain comprises theHVR-L1, HVR-L2, and HVR-L3 of the monoclonal antibody M1 produced by ahybridoma cell line with ATCC Accession Number PTA-120399 or progenythereof and/or wherein the heavy chain variable domain comprises theHVR-H1, HVR-H2, and HVR-H3 of the monoclonal antibody M1 produced by ahybridoma cell line with ATCC Accession Number PTA-120399 or progenythereof or ii) an isolated anti-C1q antibody which binds essentially thesame C1q epitope as the antibody M1 produced by the hybridoma cell linewith ATCC Accession Number PTA-120399 or anti-C1q binding fragmentsthereof. In other aspects, the present disclosure provides an anti-C1qantibody for use in treating or preventing Guillain-Barré Syndrome (GBS)in an individual, wherein the anti-C1q antibody is: i) an isolatedanti-C1q antibody comprising a light chain variable domain and a heavychain variable domain, wherein the light chain variable domain comprisesthe HVR-L1, HVR-L2, and HVR-L3 of the monoclonal antibody M1 produced bya hybridoma cell line with ATCC Accession Number PTA-120399 or progenythereof and/or wherein the heavy chain variable domain comprises theHVR-H1, HVR-H2, and HVR-H3 of the monoclonal antibody M1 produced by ahybridoma cell line with ATCC Accession Number PTA-120399 or progenythereof or ii) an isolated anti-C1q antibody which binds essentially thesame C1q epitope as the antibody M1 produced by the hybridoma cell linewith ATCC Accession Number PTA-120399 or anti-C1q binding fragmentsthereof. In other aspects, the present disclosure provides use of ananti-C1q antibody in the manufacture of a medicament for treating orpreventing Guillain-Barré Syndrome (GBS) in an individual, wherein theanti-C1q antibody is: i) an isolated anti-C1q antibody comprising alight chain variable domain and a heavy chain variable domain, whereinthe light chain variable domain comprises the HVR-L1, HVR-L2, and HVR-L3of the monoclonal antibody M1 produced by a hybridoma cell line withATCC Accession Number PTA-120399 or progeny thereof and/or wherein theheavy chain variable domain comprises the HVR-H1, HVR-H2, and HVR-H3 ofthe monoclonal antibody M1 produced by a hybridoma cell line with ATCCAccession Number PTA-120399 or progeny thereof or ii) an isolatedanti-C1q antibody which binds essentially the same C1q epitope as theantibody M1 produced by the hybridoma cell line with ATCC AccessionNumber PTA-120399 or anti-C1q binding fragments thereof.

In other aspects, the present disclosure provides a method of treatingor preventing Guillain-Barré Syndrome (GBS) in an individual, comprisingadministering to the individual a therapeutically effective amount of ananti-C1s antibody wherein the antibody is: i) an isolated anti-C1santibody comprising a light chain variable domain and a heavy chainvariable domain, wherein the light chain variable domain comprises theHVR-L1, HVR-L2, and HVR-L3 of the monoclonal antibody 5A1 produced by ahybridoma cell line with ATCC Accession Number PTA-120351, or progenythereof; ii) an isolated anti-C1s antibody comprising a light chainvariable domain and a heavy chain variable domain, wherein the heavychain variable domain comprises the HVR-H1, HVR-H2, and HVR-H3 of themonoclonal antibody 5A1 produced by a hybridoma cell line with ATCCAccession Number PTA-120351, or progeny thereof; iii) an isolatedanti-C1s antibody comprising a light chain variable domain and a heavychain variable domain, wherein the light chain variable domain comprisesthe HVR-L1, HVR-L2, and HVR-L3 and the heavy chain variable domaincomprises the HVR-H1, HVR-H2, and HVR-H3 of the monoclonal antibody 5A1produced by a hybridoma cell line with ATCC Accession Number PTA-120351or progeny thereof; iv) an isolated anti-C1s antibody comprising a lightchain variable domain and a heavy chain variable domain, wherein thelight chain variable domain comprises the HVR-L1, HVR-L2, and HVR-L3 ofthe monoclonal antibody 5C12 produced by a hybridoma cell line with ATCCAccession Number PTA-120352, or progeny thereof; v) an isolated anti-C1santibody comprising a light chain variable domain and a heavy chainvariable domain, wherein the heavy chain variable domain comprises theHVR-H1, HVR-H2, and HVR-H3 of the monoclonal antibody 5C12 produced by ahybridoma cell line with ATCC Accession Number PTA-120352, or progenythereof; vi) an isolated anti-C1s antibody comprising a light chainvariable domain and a heavy chain variable domain, wherein the lightchain variable domain comprises the HVR-L1, HVR-L2, and HVR-L3 and theheavy chain variable domain comprises the HVR-H1, HVR-H2, and HVR-H3 ofthe monoclonal antibody 5C12 produced by a hybridoma cell line with ATCCAccession Number PTA-120352, or progeny thereof; vii) an isolated murineanti-human C1s monoclonal antibody 5A1 produced by a hybridoma cell linewith ATCC Accession Number PTA-120351, or progeny thereof; viii) anisolated murine anti-human C1s monoclonal antibody 5C12 produced by ahybridoma cell line with ATCC Accession Number PTA-120352 or progenythereof; ix) an isolated anti-C1s antibody which binds essentially thesame C1s epitope as the antibody 5A1 produced by a hybridoma cell linewith ATCC Accession Number PTA-120351; or x) an isolated anti-C1santibody which binds essentially the same C1s epitope as the 5C12antibody produced by a hybridoma cell line with ATCC Accession NumberPTA-120352. In other aspects, the present disclosure provides ananti-Cis antibody for use in treating or preventing Guillain-BarréSyndrome (GBS) in an individual, wherein the anti-C1s antibody is: i) anisolated anti-C1s antibody comprising a light chain variable domain anda heavy chain variable domain, wherein the light chain variable domaincomprises the HVR-L1, HVR-L2, and HVR-L3 of the monoclonal antibody 5A1produced by a hybridoma cell line with ATCC Accession Number PTA-120351,or progeny thereof; ii) an isolated anti-C1s antibody comprising a lightchain variable domain and a heavy chain variable domain, wherein theheavy chain variable domain comprises the HVR-H1, HVR-H2, and HVR-H3 ofthe monoclonal antibody 5A1 produced by a hybridoma cell line with ATCCAccession Number PTA-120351, or progeny thereof; iii) an isolatedanti-C1s antibody comprising a light chain variable domain and a heavychain variable domain, wherein the light chain variable domain comprisesthe HVR-L1, HVR-L2, and HVR-L3 and the heavy chain variable domaincomprises the HVR-H1, HVR-H2, and HVR-H3 of the monoclonal antibody 5A1produced by a hybridoma cell line with ATCC Accession Number PTA-120351or progeny thereof; iv) an isolated anti-C1s antibody comprising a lightchain variable domain and a heavy chain variable domain, wherein thelight chain variable domain comprises the HVR-L1, HVR-L2, and HVR-L3 ofthe monoclonal antibody 5C12 produced by a hybridoma cell line with ATCCAccession Number PTA-120352, or progeny thereof; v) an isolated anti-C1santibody comprising a light chain variable domain and a heavy chainvariable domain, wherein the heavy chain variable domain comprises theHVR-H1, HVR-H2, and HVR-H3 of the monoclonal antibody 5C12 produced by ahybridoma cell line with ATCC Accession Number PTA-120352, or progenythereof; vi) an isolated anti-C1s antibody comprising a light chainvariable domain and a heavy chain variable domain, wherein the lightchain variable domain comprises the HVR-L1, HVR-L2, and HVR-L3 and theheavy chain variable domain comprises the HVR-H1, HVR-H2, and HVR-H3 ofthe monoclonal antibody 5C12 produced by a hybridoma cell line with ATCCAccession Number PTA-120352, or progeny thereof; vii) an isolated murineanti-human C1s monoclonal antibody 5A1 produced by a hybridoma cell linewith ATCC Accession Number PTA-120351, or progeny thereof; viii) anisolated murine anti-human C1s monoclonal antibody 5C12 produced by ahybridoma cell line with ATCC Accession Number PTA-120352 or progenythereof; ix) an isolated anti-C1s antibody which binds essentially thesame C1s epitope as the antibody 5A1 produced by a hybridoma cell linewith ATCC Accession Number PTA-120351; or x) an isolated anti-C1santibody which binds essentially the same C1s epitope as the 5C12antibody produced by a hybridoma cell line with ATCC Accession NumberPTA-120352. In other aspects, the present disclosure provides use of ananti-C1s antibody in the manufacture of a medicament for treating orpreventing Guillain-Barré Syndrome (GBS) in an individual, wherein theanti-C1s antibody is: i) an isolated anti-C1s antibody comprising alight chain variable domain and a heavy chain variable domain, whereinthe light chain variable domain comprises the HVR-L1, HVR-L2, and HVR-L3of the monoclonal antibody 5A1 produced by a hybridoma cell line withATCC Accession Number PTA-120351, or progeny thereof; ii) an isolatedanti-C1s antibody comprising a light chain variable domain and a heavychain variable domain, wherein the heavy chain variable domain comprisesthe HVR-H1, HVR-H2, and HVR-H3 of the monoclonal antibody 5A1 producedby a hybridoma cell line with ATCC Accession Number PTA-120351, orprogeny thereof; iii) an isolated anti-C1s antibody comprising a lightchain variable domain and a heavy chain variable domain, wherein thelight chain variable domain comprises the HVR-L1, HVR-L2, and HVR-L3 andthe heavy chain variable domain comprises the HVR-H1, HVR-H2, and HVR-H3of the monoclonal antibody 5A1 produced by a hybridoma cell line withATCC Accession Number PTA-120351 or progeny thereof; iv) an isolatedanti-C1s antibody comprising a light chain variable domain and a heavychain variable domain, wherein the light chain variable domain comprisesthe HVR-L1, HVR-L2, and HVR-L3 of the monoclonal antibody 5C12 producedby a hybridoma cell line with ATCC Accession Number PTA-120352, orprogeny thereof; v) an isolated anti-C1s antibody comprising a lightchain variable domain and a heavy chain variable domain, wherein theheavy chain variable domain comprises the HVR-H1, HVR-H2, and HVR-H3 ofthe monoclonal antibody 5C12 produced by a hybridoma cell line with ATCCAccession Number PTA-120352, or progeny thereof; vi) an isolatedanti-C1s antibody comprising a light chain variable domain and a heavychain variable domain, wherein the light chain variable domain comprisesthe HVR-L1, HVR-L2, and HVR-L3 and the heavy chain variable domaincomprises the HVR-H1, HVR-H2, and HVR-H3 of the monoclonal antibody 5C12produced by a hybridoma cell line with ATCC Accession Number PTA-120352,or progeny thereof; vii) an isolated murine anti-human C1s monoclonalantibody 5A1 produced by a hybridoma cell line with ATCC AccessionNumber PTA-120351, or progeny thereof; viii) an isolated murineanti-human C1s monoclonal antibody 5C12 produced by a hybridoma cellline with ATCC Accession Number PTA-120352 or progeny thereof; ix) anisolated anti-C1s antibody which binds essentially the same C1s epitopeas the antibody 5A1 produced by a hybridoma cell line with ATCCAccession Number PTA-120351; or x) an isolated anti-C1s antibody whichbinds essentially the same C1s epitope as the 5C12 antibody produced bya hybridoma cell line with ATCC Accession Number PTA-120352. In someembodiments, an anti-C1s antibody of this disclosure specifically bindsto and neutralizes a biological activity of C1s or the C1s proenzyme. Incertain embodiments, the biological activity is C1s binding to C1q, C1sbinding to C1r, or C1s binding to C2 or C4. In certain embodiments, thebiological activity is the proteolytic enzyme activity of C1s, theconversion of the C1s proenzyme to an active protease, or proteolyticcleavage of C4. In certain embodiments, the biological activity isactivation of the classical complement activation pathway, activation ofantibody and complement dependent cytotoxicity, or C1F hemolysis.

In certain embodiments that may be combined with any of the precedingembodiments, the individual has GBS. In certain embodiments that may becombined with any of the preceding embodiments, the individual is ahuman. In certain embodiments that may be combined with any of thepreceding embodiments, the antibody binds C1q, C1r, or C1s. In certainembodiments that may be combined with any of the preceding embodiments,the antibody is an anti-C1q antibody. In certain embodiments that may becombined with any of the preceding embodiments, the antibody is anisolated anti-C1q antibody, which binds to a C1q protein and binds toone or more amino acids of the C1q protein within amino acid residuesselected from the group consisting of: i) amino acid residues 196-226 ofSEQ ID NO:1, or amino acid residues of a C1q protein chain A (C1qA)corresponding to amino acid residues 196-226(GLFQVVSGGMVLQLQQGDQVWVEKDPKKGHI) of SEQ ID NO:1; ii) amino acidresidues 196-221 of SEQ ID NO:1, or amino acid residues of a C1qAcorresponding to amino acid residues 196-221(GLFQVVSGGMVLQLQQGDQVWVEKDP) of SEQ ID. NO:1; iii) amino acid residues202-221 of SEQ ID NO:1, or amino acid residues of a C1qA correspondingto amino acid residues 202-221 (SGGMVLQLQQGDQVWVEKD) of SEQ ID NO:1; iv)amino acid residues 202-219 of SEQ ID NO:1, or amino acid residues of aC1qA corresponding to amino acid residues 202-219 (SGGMVLQLQQGDQVWVEK)of SEQ ID NO:1; and v) amino acid residues Lys 219 and/or Ser 202 of SEQID NO:1, or amino acid residues of a C1qA corresponding Lys 219 and/orSer 202 of SEQ ID NO:1. In certain embodiments that may be combined withany of the preceding embodiments, the anti-C1q antibody further binds toone or more amino acids of the C1q protein within amino acid residuesselected from the group consisting of: i) amino acid residues 218-240 ofSEQ ID NO:3 or amino acid residues of a C1q protein chain C (C1qC)corresponding to amino acid residues 218-240 (WLAVNDYYDMVGI QGSDSVFSGF)of SEQ ID NO:3; amino acid residues 225-240 of SEQ ID NO:3 or amino acidresidues of a C1qC corresponding to amino acid residues 225-240 (YDMVGIQGSDSVFSGF) of SEQ ID NO:3; iii) amino acid residues 225-232 of SEQ IDNO:3 or amino acid residues of a C1qC corresponding to amino acidresidues 225-232 (YDMVGIQG) of SEQ ID NO:3; iv) amino acid residue Tyr225 of SEQ ID NO:3 or an amino acid residue of a C1qC corresponding toamino acid residue Tyr 225 of SEQ ID NO:3; v) amino acid residues174-196 of SEQ ID NO:3 or amino acid residues of a C1qC corresponding toamino acid residues 174-196 (HTANLCVLLYRSGVKVVTFCGHT) of SEQ ID NO:3;vi) amino acid residues 184-192 of SEQ ID NO:3 or amino acid residues ofa C1qC corresponding to amino acid residues 184-192 (RSGVKVVTF) of SEQID NO:3; vii) amino acid residues 185-187 of SEQ ID NO:3 or amino acidresidues of a C1qC corresponding to amino acid residues 185-187 (SGV) ofSEQ ID NO:3; and viii) amino acid residue Ser 185 of SEQ ID NO:3 or anamino acid residue of a C1qC corresponding to amino acid residue Ser 185of SEQ ID NO:3.

In certain embodiments that may be combined with any of the precedingembodiments, the antibody is an isolated anti-C1q antibody, which bindsto a C1q protein and binds to one or more amino acids of the C1q proteinchain A (C1qA) within amino acid residues selected from the groupconsisting of: i) amino acid residues 196-226 of SEQ ID NO:1, or aminoacid residues of a C1q protein chain A (C1qA) corresponding to aminoacid residues 196-226 (GLFQVVSGGMVLQLQQGDQVWVEKDPKKGHI) of SEQ ID NO:1;ii) amino acid residues 196-221 of SEQ ID NO:1, or amino acid residuesof a C1qA corresponding to amino acid residues 196-221(GLFQVVSGGMVLQLQQGDQVWVEKDP) of SEQ ID. NO:1; iii) amino acid residues202-221 of SEQ ID NO:1, or amino acid residues of a C1qA correspondingto amino acid residues 202-221 (SGGMVLQLQQGDQVWVEKD) of SEQ ID NO:1; iv)amino acid residues 202-219 of SEQ ID NO:1, or amino acid residues of aC1qA corresponding to amino acid residues 202-219 (SGGMVLQLQQGDQVWVEK)of SEQ ID NO:1; and v) amino acid residue Lys 219 of SEQ ID NO:1, or anamino acid residue of a C1qA corresponding Lys 219 of SEQ ID NO:1; andwherein the isolated anti-C1q antibody binds to one or more amino acidsof the C1q protein chain C (C1qC) within amino acid residues selectedfrom the group consisting of: i) amino acid residues 174-196 of SEQ IDNO:3 or amino acid residues of a C1qC corresponding to amino acidresidues 174-196 (HTANLCVLLYRSGVKVVTFCGHT) of SEQ ID NO:3; ii) aminoacid residues 184-192 of SEQ ID NO:3 or amino acid residues of a C1qCcorresponding to amino acid residues 184-192 (RSGVKVVTF) of SEQ ID NO:3;iii) amino acid residues 185-187 of SEQ ID NO:3 or amino acid residuesof a C1qC corresponding to amino acid residues 185-187 (SGV) of SEQ IDNO:3; and iv) amino acid residue Ser 185 of SEQ ID NO:3 or an amino acidresidue of a C1qC corresponding to amino acid residue Ser 185 of SEQ IDNO:3. In certain embodiments that may be combined with any of thepreceding embodiments, the antibody is an anti-C1q antibody, wherein theanti-C1q antibody binds specifically to both human C1q and mouse C1q. Incertain embodiments that may be combined with any of the precedingembodiments, the antibody is an anti-C1q antibody, wherein the anti-C1qantibody has dissociation constant (K_(D)) for human C1q and mouse C1qof less than 100 pM. In certain embodiments that may be combined withany of the preceding embodiments, the antibody is an anti-Cq1q antibody,wherein the anti-C1q antibody specifically binds to and neutralizes abiological activity of C1q. In certain embodiments that may be combinedwith any of the preceding embodiments, the antibody is an anti-C1qantibody, wherein the biological activity is (1) C1q binding to anautoantibody, (2) C1q binding to C1r, (3) C1q binding to C1s, (4) C1qbinding to phosphatidylserine, (5) C1q binding to pentraxin-3, (6) C1qbinding to C-reactive protein (CRP), (7) C1q binding to globular C1qreceptor (gC1qR), (8) C1q binding to complement receptor 1 (CR1), (9)C1q binding to beta-amyloid, or (10) C1q binding to calreticulin. Incertain embodiments that may be combined with any of the precedingembodiments, the antibody is an anti-C1q antibody, wherein thebiological activity is (1) activation of the classical complementactivation pathway, (2) activation of antibody and complement dependentcytotoxicity, (3) CH50 hemolysis, (4) synapse loss, (5) B-cell antibodyproduction, (6) dendritic cell maturation, (7) T-cell proliferation, (8)cytokine production (9) microglia activation, (10) Arthus reaction, (11)phagocytosis of synapses or nerve endings, or (12) activation ofcomplement receptor 3 (CR3/C3) expressing cells. In certain embodimentsthat may be combined with any of the preceding embodiments, the antibodyis an anti-C1s antibody. In certain embodiments that may be combinedwith any of the preceding embodiments, the antibody is an anti-C1santibody, wherein the anti-C1s antibody specifically binds to andneutralizes a biological activity of C1s or the C1s proenzyme. Incertain embodiments that may be combined with any of the precedingembodiments, the antibody is an anti-C1s antibody, wherein saidbiological activity is (1) C1s binding to C1q, (2) C1s binding to C1r,or (3) C1s binding to C2 or C4. In certain embodiments that may becombined with any of the preceding embodiments, the antibody is ananti-C1s antibody, wherein said biological activity is (1) theproteolytic enzyme activity of C1s, (2) the conversion of the C1sproenzyme to an active protease, or (3) cleavage of C4. In certainembodiments that may be combined with any of the preceding embodiments,the antibody is an anti-C1s antibody, wherein said biological activityis (1) activation of the classical complement activation pathway, (2)activation of antibody and complement dependent cytotoxicity, or (3) C1Fhemolysis. In certain embodiments that may be combined with any of thepreceding embodiments, the antibody is an anti-C1s antibody, wherein theanti-C1s antibody is a murine antibody. In certain embodiments that maybe combined with any of the preceding embodiments, the antibody is ananti-C1s antibody, wherein said antibody is capable of neutralizing atleast 30%, at least 50%, or at least 70% of C1F hemolysis. In certainembodiments that may be combined with any of the preceding embodiments,the antibody is an anti-C1r antibody. In certain embodiments that may becombined with any of the preceding embodiments, the antibody is ananti-C1 complex antibody. In certain embodiments that may be combinedwith any of the preceding embodiments, the antibody binds human C1q,C1r, or C1s. In certain embodiments that may be combined with any of thepreceding embodiments, the antibody binds human C1 complex. In certainembodiments that may be combined with any of the preceding embodiments,the antibody is a monoclonal antibody. In certain embodiments that maybe combined with any of the preceding embodiments, the antibody is amouse antibody, a human antibody, a humanized antibody, or a chimericantibody. In certain embodiments that may be combined with any of thepreceding embodiments, the antibody is of the IgG class, including IgG₁,IgG₂, IgG₃, or IgG₄ isotypes. In certain embodiments that may becombined with any of the preceding embodiments, the antibody is anantibody fragment selected from the group consisting of Fab, Fab′-SH,Fv, scFv, and (Fab′)₂ fragments. In certain embodiments that may becombined with any of the preceding embodiments, the antibody is abispecific antibody recognizing a first antigen and a second antigen. Incertain embodiments that may be combined with any of the precedingembodiments, the first antigen is selected from the group consisting ofC1q, C1r, C1s, and the C1 complex and the second antigen is an antigenfacilitating transport across the blood-brain-barrier. In certainembodiments that may be combined with any of the preceding embodiments,the second antigen is selected from the group consisting of transferrinreceptor (TR), insulin receptor (HIR), insulin-like growth factorreceptor (IGFR), low-density lipoprotein receptor related proteins 1 and2 (LPR-1 and 2), diphtheria toxin receptor, CRM197, a llama singledomain antibody, TMEM 30(A), a protein transduction domain, TAT, Syn-B,penetratin, a poly-arginine peptide, an angiopep peptide, and ANG1005.In certain embodiments that may be combined with any of the precedingembodiments, the antibody inhibits C3c deposition. In certainembodiments that may be combined with any of the preceding embodiments,the antibody inhibits membrane attack complex (MAC) deposition. Incertain embodiments that may be combined with any of the precedingembodiments, the antibody inhibits axonal damage formation. In certainembodiments that may be combined with any of the preceding embodiments,the antibody inhibits respiratory muscle damage. In certain embodimentsthat may be combined with any of the preceding embodiments, the antibodyinhibits the classical complement activation pathway by an amount thatranges from at least 30% to at least 99.9%. In certain embodiments thatmay be combined with any of the preceding embodiments, the antibodyinhibits the alternative complement activation pathway by an amount thatranges from at least 30% to at least 99.9%. In certain embodiments thatmay be combined with any of the preceding embodiments, the antibodyinhibits complement-dependent cell-mediated cytotoxicity (CDCC)activation pathway by an amount that ranges from at least 30% to atleast 99.9%. In certain embodiments that may be combined with any of thepreceding embodiments, the antibody does not inhibit the lectincomplement activation pathway. In certain embodiments that may becombined with any of the preceding embodiments, the antibody comprises adissociation constant (K_(D)) for its corresponding antigen that rangesfrom 100 nM to 0.005 nM or less than 0.005 nM. In certain embodimentsthat may be combined with any of the preceding embodiments, the antibodyinhibits autoantibody-dependent and complement-dependent cytotoxicity(CDC). In certain embodiments that may be combined with any of thepreceding embodiments, the antibody prevents amplification of thealternative complement activation pathway initiated by C1q binding. Incertain embodiments that may be combined with any of the precedingembodiments, the antibody comprises an EC₅₀ that ranges from 3 μg/ml to0.05 μg/ml, or less than 0.05 μg/ml. In certain embodiments that may becombined with any of the preceding embodiments, the antibody does notinhibit autoantibody-dependent cellular cytotoxicity (ADCC). In certainembodiments that may be combined with any of the preceding embodiments,the method further comprises administering to the individual atherapeutically effective amount of a second antibody, wherein thesecond antibody is selected from the group consisting of the anti-C1qantibody, the anti-C1r antibody, the anti-C1s antibody and the anti-C1complex antibody. In certain embodiments that may be combined with anyof the preceding embodiments, the method further comprises administeringto the individual a therapeutically effective amount of an inhibitor ofantibody-dependent cellular cytotoxicity (ADCC). In certain embodimentsthat may be combined with any of the preceding embodiments, the methodfurther comprises administering to the individual a therapeuticallyeffective amount of an inhibitor of the alternative complementactivation pathway. In certain embodiments that may be combined with anyof the preceding embodiments, the method further comprises administeringto the individual an inhibitor of the interaction between theautoantibody and an autoantigen. In certain embodiments that may becombined with any of the preceding embodiments, the antibody binds itscorresponding antigen with a binding stoichiometry that ranges from 20:1to 1.0:1 or less than 1.0:1.

In other aspects, the present disclosure provides a diagnostic kitcomprising an antibody of any of the preceding embodiments for treatingor preventing Guillain-Barré Syndrome (GBS) in an individual.

It is to be understood that one, some, or all of the properties of thevarious embodiments described herein may be combined to form otherembodiments of the compositions and methods provided herein. These andother aspects of the compositions and methods provided herein willbecome apparent to one of skill in the art.

DESCRIPTION OF THE FIGURES

FIG. 1 illustrates the results of an ELISA screen for antibodiesspecifically binding human C1s or human C1s proenzyme. The bindingassays were conducted either in the absence of an anti-C1s antibody(“media”) or in the presence of one of six anti-C1s antibodies (1B4,3F8, 3G3, 5A1, 5C12, or 7C4). Left columns show binding signals foranti-C1s antibody binding to the C1s protein; middle columns showbinding signals for anti-C1s antibody binding to the C1s proenzyme;right columns show binding signals for anti-C1s antibody binding to thehuman transferrin (HT) negative control protein.

FIG. 2 illustrates the C1s neutralizing activities of anti-C1santibodies in a C1F hemolytic assay. FIG. 2A illustrates the results ofassays conducted with six anti-C1s antibodies (1B4, 3F8, 3G3, 5A1, 5C12,or 7C4) in a single-dose format. FIG. 2B shows the results of C1Fhemolytic assays conducted with two anti-C1s antibodies (5A1 and 5C12)in a dose-response format.

FIG. 3 illustrates the C1s neutralizing activities of anti-C1santibodies in a C4 cleavage assay. The upper panel illustrates theactivity of eight anti-C1s antibodies (M241, 3F8, 3A1, 3A2, 2A1, 5C12,6HK, and 8HK) regarding the inhibition of C4 cleavage at a singleconcentration. The lower panel illustrates the neutralizing activitiesof two anti-C1s antibodies (5A1 and 5C12) in a dose-response format.

FIG. 4 illustrates mass spectrometry characterization of C1q antibodycomplexes. FIG. 4A depicts a mixture of ANN-001 (4A4B11) and C1q showingthat ANN-001 monomer at the predicted mass of ˜150 kDa, C1q monomer atthe expected mass of ˜460 kDa, and the C1q/ANN-001 1:1 complex at thepredicted mass of ˜600 kDa. FIG. 4B depicts a mixture of ANN-005 (M1)and C1q showing that ANN-005 monomer at the predicted mass of ˜150 kDa,C1q monomer at the expected mass of ˜460 kDa, and the C1q/ANN-005 1:1complex at the predicted mass of ˜600 kDa.

FIG. 5A shows a general schematic representation of the complementcascade, including the three complement activation pathways and theterminal pathway. FIG. 5B shows a schematic of the C1 complex. The C1sand C1r dimers are seen in a complex with the C1q hexamer.

FIG. 6 illustrates how anti-C1 antibodies can be used to preventGBS-anti-ganglioside antibody dependent complement deposition ondiaphragm motor nerve terminals ex vivo. Whole-mount muscles maintainedalive in oxygenated Ringer's solution were incubated with theanti-ganglioside monoclonal antibody CGM3 (50 μg/ml) for 2-2.5 h at 32°C., then for 30 min at 4° C. and then equilibrated for 10 min at roomtemperature, rinsed in Ringer's medium and subsequently exposed to 40%Normal Human Serum (NHS) as a source of complement in Ringer's mediumfor 1 h at room temperature. Anti-C1 antibodies (100 μg/ml) or thecontrol mAb (100 μg/ml) were mixed with NHS 10 min prior to theincubation of the muscle preparation. C3c was detected by incubationwith FITC-labelled rabbit anti-C3c (1/300; Dako, Ely, UK) for 1 h at 4°C., and nerve terminals detected by incubation with TRITC labelledα-bungarotoxin (BTx; 1/750; Sigma, UK) that binds nicotinicacetylcholine receptors (nAChR) on the postsynaptic membrane of theneuromuscular junction (NMJ). Digital images were captured using both aZeiss Pascal confocal laser scanning microscope and a Zeiss Axio ImagerZ1 with ApoTome. Image-analysis measurements were made using ImageJ(NIH) image analysis software. For quantitative analysis of C3c threestaining runs were performed on tissue from at least three individualmuscle preparations, and quantified as previously described (O'Hanlon etal., 2001) and displayed as box and whisker graphs.

FIG. 7 illustrates that anti-C1q antibodies can suppress complementdeposition and preserve axonal integrity in an ex-vivo GBS assay. FIG.7A shows a quantitative representation of the immunofluorescent labelingof C3c deposition on the explanted diaphragm plotted as a box andwhisker plot to represent the spread of the nonparametric data. FIG. 7Bdepicts images of sections quantitated in FIG. 3A showing TRITC-labelledBTx staining of the nAChR in the top panel, FITC-labelled rabbitanti-C3c labeling in the middle panel, and the merged images in thebottom panel. The tissues are either untreated (no complement added) ortreated with a control IgG1 antibody or the anti-C1q antibodies 4A4B11and M1. FIG. 7C shows a quantitative representation of the amount ofaxonal neurofilament labeled by the rabbit polyclonal serum 1211. FIG.7D depicts representative images of NMJ showing the post-synapticmembrane (nAChR) and axon (Nfil) staining in the presence of theanti-C1q and control antibodies.

FIG. 8 shows that the anti-C1q antibody M1 can prevent complementdeposition and axonal degradation in an in vivo mouse model of GBS.Balb/c mice (3-4 weeks old, 10-15 g) were injected intraperitoneallywith 1.5 mg CGM3, followed 16 h later by an intravascular (i.v.)injection of 200 μg of anti-C1q M1 antibody and intraperitonealinjection of 0.5 ml 100% NHS. After 4 hours the mice were euthanized anddiaphragm muscle tissue was dissected and processed forimmunohistological analyses. FIG. 8A shows a box and whisker plot of thequantitation of the C3c immunofluorescence at the motor nerve endplateand corresponding images of C3c (green) deposition at the NMJ withpost-synaptic membrane on the muscle fluorescently labeled by (BTx red)from each treatment group. FIG. 8B shows a box and whisker plot of thequantitation of the MAC immunofluorescence at the motor nerve endplateand below are images of MAC (green) deposition at the NMJ with musclefluorescently labeled by BTx (red). FIG. 8C shows quantitation of theneurofilament staining at the NMJ and below corresponding images of theneurofilament staining.

FIG. 9A shows baseline tidal volume readings recorded the day beforetreatment with anti-C1q antibody M1 or isotype control antibody ACP1 andsubsequent readings were taken at 4 h and 6 h post-injury induction.FIG. 9B shows the percentage change from baseline tidal volume for theexperimental groups treated with anti-C1q antibody M1 or isotype controlantibody ACP1.

DETAILED DESCRIPTION OF THE INVENTION General Techniques

The techniques and procedures described or referenced herein aregenerally well understood and commonly employed using conventionalmethodology by those skilled in the art, such as, for example, thewidely utilized methodologies described in Sambrook et al., MolecularCloning: A Laboratory Manual 3d edition (2001) Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.; Current Protocols inMolecular Biology (F. M. Ausubel, et al. eds., (2003)); the seriesMethods in Enzymology (Academic Press, Inc.): PCR 2: A PracticalApproach (M. J. MacPherson, B. D. Hames and G. R. Taylor eds. (1995)),Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and AnimalCell Culture (R. I. Freshney, ed. (1987)); Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; CellBiology: A Laboratory Notebook (J. E. Cellis, ed., 1998) Academic Press;Animal Cell Culture (R. I. Freshney), ed., 1987); Introduction to Celland Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press;Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B.Griffiths, and D. G. Newell, eds., 1993-8) J. Wiley and Sons; Handbookof Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.); GeneTransfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos,eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds.,1994); Current Protocols in Immunology (J. E. Coligan et al., eds.,1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999);Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P.Finch, 1997); Antibodies: A Practical Approach (D. Catty, ed., IRLPress, 1988-1989); Monoclonal Antibodies: A Practical Approach (P.Shepherd and C. Dean, eds., Oxford University Press, 2000); UsingAntibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold SpringHarbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D.Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principlesand Practice of Oncology (V. T. DeVita et al., eds., J.B. LippincottCompany, 1993).

DEFINITIONS

As used herein, the term “preventing” includes providing prophylaxiswith respect to occurrence or recurrence of a particular disease,disorder, or condition in an individual. An individual may bepredisposed to, susceptible to a particular disease, disorder, orcondition, or at risk of developing such a disease, disorder, orcondition, but has not yet been diagnosed with the disease, disorder, orcondition.

As used herein, an individual “at risk” of developing a particulardisease, disorder, or condition may or may not have detectable diseaseor symptoms of disease, and may or may not have displayed detectabledisease or symptoms of disease prior to the treatment methods describedherein. “At risk” denotes that an individual has one or more riskfactors, which are measurable parameters that correlate with developmentof a particular disease, disorder, or condition, as known in the art. Anindividual having one or more of these risk factors has a higherprobability of developing a particular disease, disorder, or conditionthan an individual without one or more of these risk factors.

As used herein, the term “treatment” refers to clinical interventiondesigned to alter the natural course of the individual being treatedduring the course of clinical pathology. Desirable effects of treatmentinclude decreasing the rate of progression, ameliorating or palliatingthe pathological state, and remission or improved prognosis of aparticular disease, disorder, or condition. An individual issuccessfully “treated”, for example, if one or more symptoms associatedwith a particular disease, disorder, or condition are mitigated oreliminated.

An “effective amount” refers to at least an amount effective, at dosagesand for periods of time necessary, to achieve the desired therapeutic orprophylactic result. An effective amount can be provided in one or moreadministrations.

A “therapeutically effective amount” is at least the minimumconcentration required to effect a measurable improvement of aparticular disease, disorder, or condition. A therapeutically effectiveamount herein may vary according to factors such as the disease state,age, sex, and weight of the patient, and the ability of the anti-C1q,anti-C1s, anti-C1r, and/or anti-C1 complex antibody to elicit a desiredresponse in the individual. A therapeutically effective amount is alsoone in which any toxic or detrimental effects of the anti-C1q, anti-C1s,anti-C1r, and/or anti-C1 complex antibody are outweighed by thetherapeutically beneficial effects.

“Chronic” administration refers to administration of the medicament(s)in a continuous as opposed to acute mode, so as to maintain the initialtherapeutic effect (activity) for an extended period of time.“Intermittent” administration refers to treatment that is notconsecutively done without interruption, but rather is cyclic in nature.

As used herein, administration “in conjunction” with another compound orcomposition includes simultaneous administration and/or administrationat different times. Administration in conjunction also encompassesadministration as a co-formulation or administration as separatecompositions, including at different dosing frequencies or intervals,and using the same route of administration or different routes ofadministration.

An “individual” for purposes of treatment, prevention, or reduction ofrisk refers to any animal classified as a mammal, including humans,domestic and farm animals, and zoo, sport, or pet animals, such as dogs,horses, rabbits, cattle, pigs, hamsters, gerbils, mice, ferrets, rats,cats, and the like. Preferably, the individual is human.

As used herein, “autoantibody” means any antibody that recognizes a hostantigen, such as AQP4, in an individual having GBS and activates theclassical pathway of complement activation. In the first step of thisactivation process complement factor C1q binds to theautoantibody-autoantigen-immune complex. Autoantibodies may includenaturally occurring antibodies, such as serum antibodies from GBSpatients (commonly referred to as GBS-IgG) or monoclonal antibodies,such as rAb-53.

The term “immunoglobulin” (Ig) is used interchangeably with “antibody”herein. The term “antibody” herein is used in the broadest sense andspecifically covers monoclonal antibodies, polyclonal antibodies,multispecific antibodies (e.g. bispecific antibodies) formed from atleast two intact antibodies, and antibody fragments so long as theyexhibit the desired biological activity.

The basic 4-chain antibody unit is a heterotetrameric glycoproteincomposed of two identical light (L) chains and two identical heavy (H)chains. The pairing of a V_(H) and V_(L) together forms a singleantigen-binding site. For the structure and properties of the differentclasses of antibodies, see, e.g., Basic and Clinical Immunology, 8thEd., Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.),Appleton & Lange, Norwalk, Conn., 1994, page 71 and

Chapter 6.

The L chain from any vertebrate species can be assigned to one of twoclearly distinct types, called kappa (“κ”) and lambda (“λ”), based onthe amino acid sequences of their constant domains. Depending on theamino acid sequence of the constant domain of their heavy chains (CH),immunoglobulins can be assigned to different classes or isotypes. Thereare five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, havingheavy chains designated alpha (“α”), delta (“δ”), epsilon (“ε”), gamma(“γ”) and mu (“μ”), respectively. The γ and α classes are furtherdivided into subclasses (isotypes) on the basis of relatively minordifferences in the CH sequence and function, e.g., humans express thefollowing subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. Thesubunit structures and three dimensional configurations of differentclasses of immunoglobulins are well known and described generally in,for example, Abbas et al., Cellular and Molecular Immunology, 4^(th) ed.(W.B. Saunders Co., 2000).

“Native antibodies” are usually heterotetrameric glycoproteins of about150,000 daltons, composed of two identical light (L) chains and twoidentical heavy (H) chains. Each light chain is linked to a heavy chainby one covalent disulfide bond, while the number of disulfide linkagesvaries among the heavy chains of different immunoglobulin isotypes. Eachheavy and light chain also has regularly spaced intrachain disulfidebridges. Each heavy chain has at one end a variable domain (V_(H))followed by a number of constant domains. Each light chain has avariable domain at one end (V_(L)) and a constant domain at its otherend; the constant domain of the light chain is aligned with the firstconstant domain of the heavy chain, and the light chain variable domainis aligned with the variable domain of the heavy chain. Particular aminoacid residues are believed to form an interface between the light chainand heavy chain variable domains.

An “isolated” antibody, such as an anti-C1q, anti-C1s, anti-C1r, and/oranti-C1 complex antibody of the present disclosure, is one that has beenidentified, separated and/or recovered from a component of itsproduction environment (e.g., naturally or recombinantly). Preferably,the isolated polypeptide is free of association with all othercontaminant components from its production environment. Contaminantcomponents from its production environment, such as those resulting fromrecombinant transfected cells, are materials that would typicallyinterfere with research, diagnostic or therapeutic uses for theantibody, and may include enzymes, hormones, and other proteinaceous ornon-proteinaceous solutes. In preferred embodiments, the polypeptidewill be purified: (1) to greater than 95% by weight of antibody asdetermined by, for example, the Lowry method, and in some embodiments,to greater than 99% by weight; (2) to a degree sufficient to obtain atleast 15 residues of N-terminal or internal amino acid sequence by useof a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE undernon-reducing or reducing conditions using Coomassie blue or, preferably,silver stain. Isolated antibody includes the antibody in situ withinrecombinant T-cells since at least one component of the antibody'snatural environment will not be present. Ordinarily, however, anisolated polypeptide or antibody will be prepared by at least onepurification step.

The “variable region” or “variable domain” of an antibody, such as ananti-C1q, anti-C1s, anti-C1r, and/or anti-C1 complex antibody of thepresent disclosure, refers to the amino-terminal domains of the heavy orlight chain of the antibody. The variable domains of the heavy chain andlight chain may be referred to as “V_(H)” and “V_(L)”, respectively.These domains are generally the most variable parts of the antibody(relative to other antibodies of the same class) and contain the antigenbinding sites.

The term “variable” refers to the fact that certain segments of thevariable domains differ extensively in sequence among antibodies, suchas anti-C1q, anti-C1s, anti-C1r, and/or anti-C1 complex antibodies ofthe present disclosure. The V domain mediates antigen binding anddefines the specificity of a particular antibody for its particularantigen. However, the variability is not evenly distributed across theentire span of the variable domains. Instead, it is concentrated inthree segments called hypervariable regions (HVRs) both in thelight-chain and the heavy chain variable domains. The more highlyconserved portions of variable domains are called the framework regions(FR). The variable domains of native heavy and light chains eachcomprise four FR regions, largely adopting a beta-sheet configuration,connected by three HVRs, which form loops connecting, and in some casesforming part of, the beta-sheet structure. The HVRs in each chain areheld together in close proximity by the FR regions and, with the HVRsfrom the other chain, contribute to the formation of the antigen bindingsite of antibodies (see Kabat et al., Sequences of ImmunologicalInterest, Fifth Edition, National Institute of Health, Bethesda, Md.(1991)). The constant domains are not involved directly in the bindingof antibody to an antigen, but exhibit various effector functions, suchas participation of the antibody in antibody-dependent-cellulartoxicity.

The term “monoclonal antibody” as used herein refers to an antibody,such as an anti-C1q, anti-C1s, anti-C1r, and/or anti-C1 complex antibodyof the present disclosure, obtained from a population of substantiallyhomogeneous antibodies, i.e., the individual antibodies comprising thepopulation are identical except for possible naturally occurringmutations and/or post-translation modifications (e.g., isomerizations,amidations) that may be present in minor amounts. Monoclonal antibodiesare highly specific, being directed against a single antigenic site. Incontrast to polyclonal antibody preparations which typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen. In addition to their specificity, the monoclonal antibodies areadvantageous in that they are synthesized by the hybridoma culture,uncontaminated by other immunoglobulins. The modifier “monoclonal”indicates the character of the antibody as being obtained from asubstantially homogeneous population of antibodies, and is not to beconstrued as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies to be used in accordancewith the present invention may be made by a variety of techniques,including, for example, the hybridoma method (e.g., Kohler and Milstein,Nature, 256:495-97 (1975); Hongo et al., Hybridoma, 14 (3):253-260(1995), Harlow et al., Antibodies: A Laboratory Manual, (Cold SpringHarbor Laboratory Press, 2d ed. 1988); Hammerling et al., in: MonoclonalAntibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981)),recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567),phage-display technologies (see, e.g., Clackson et al., Nature,352:624-628 (1991); Marks et al., J. Mol. Biol. 222:581-597 (1992);Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol.Biol. 340(5):1073-1093 (2004); Fellouse, Proc. Nat'l Acad. Sci. USA101(34):12467-472 (2004); and Lee et al., J. Immunol. Methods284(1-2):119-132 (2004), and technologies for producing human orhuman-like antibodies in animals that have parts or all of the humanimmunoglobulin loci or genes encoding human immunoglobulin sequences(see, e.g., WO 1998/24893; WO 1996/34096; WO 1996/33735; WO 1991/10741;Jakobovits et al., Proc. Nat'l Acad. Sci. USA 90:2551 (1993); Jakobovitset al., Nature 362:255-258 (1993); Bruggemann et al., Year in Immunol.7:33 (1993); U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126;5,633,425; and U.S. Pat. No. 5,661,016; Marks et al., Bio/Technology10:779-783 (1992); Lonberg et al., Nature 368:856-859 (1994); Morrison,Nature 368:812-813 (1994); Fishwild et al., Nature Biotechnol.14:845-851 (1996); Neuberger, Nature Biotechnol. 14:826 (1996); andLonberg and Huszar, Intern. Rev. Immunol. 13:65-93 (1995).

The terms “full-length antibody,” “intact antibody” or “whole antibody”are used interchangeably to refer to an antibody, such as an anti-C1q,anti-C1s, anti-C1r, and/or anti-C1 complex antibody of the presentdisclosure, in its substantially intact form, as opposed to an antibodyfragment. Specifically whole antibodies include those with heavy andlight chains including an Fc region. The constant domains may be nativesequence constant domains (e.g., human native sequence constant domains)or amino acid sequence variants thereof. In some cases, the intactantibody may have one or more effector functions.

An “antibody fragment” comprises a portion of an intact antibody,preferably the antigen binding and/or the variable region of the intactantibody. Examples of antibody fragments include Fab, Fab′, F(ab′)₂ andFv fragments; diabodies; linear antibodies (see U.S. Pat. No. 5,641,870,Example 2; Zapata et al., Protein Eng. 8(10):1057-1062 (1995));single-chain antibody molecules and multispecific antibodies formed fromantibody fragments.

Papain digestion of antibodies, such as anti-C1q, anti-C1s, anti-C1r,and/or anti-C1 complex antibodies of the present disclosure, producestwo identical antigen-binding fragments, called “Fab” fragments, and aresidual “Fc” fragment, a designation reflecting the ability tocrystallize readily. The Fab fragment consists of an entire L chainalong with the variable region domain of the H chain (V_(H)), and thefirst constant domain of one heavy chain (C_(H)1). Each Fab fragment ismonovalent with respect to antigen binding, i.e., it has a singleantigen-binding site. Pepsin treatment of an antibody yields a singlelarge F(ab′)₂ fragment which roughly corresponds to two disulfide linkedFab fragments having different antigen-binding activity and is stillcapable of cross-linking antigen. Fab′ fragments differ from Fabfragments by having a few additional residues at the carboxy terminus ofthe C_(H)1 domain including one or more cysteines from the antibodyhinge region. Fab′-SH is the designation herein for Fab′ in which thecysteine residue(s) of the constant domains bear a free thiol group.F(ab′)₂ antibody fragments originally were produced as pairs of Fab′fragments which have hinge cysteines between them. Other chemicalcouplings of antibody fragments are also known.

The Fc fragment comprises the carboxy-terminal portions of both H chainsheld together by disulfides. The effector functions of antibodies aredetermined by sequences in the Fc region, the region which is alsorecognized by Fc receptors (FcR) found on certain types of cells.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and -binding site. This fragment consists of a dimerof one heavy- and one light-chain variable region domain in tight,non-covalent association. From the folding of these two domains emanatesix hypervariable loops (3 loops each from the H and L chain) thatcontribute the amino acid residues for antigen binding and conferantigen binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three HVRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

“Single-chain Fv” also abbreviated as “sFv” or “scFv” are antibodyfragments that comprise the VH and VL antibody domains connected into asingle polypeptide chain. Preferably, the sFv polypeptide furthercomprises a polypeptide linker between the V_(H) and V_(L) domains whichenables the sFv to form the desired structure for antigen binding. For areview of the sFv, see Plückthun in The Pharmacology of MonoclonalAntibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, NewYork, pp. 269-315 (1994).

“Functional fragments” of antibodies, such as anti-C1q, anti-C1s,anti-C1r, and/or anti-C1 complex antibodies of the present disclosure,comprise a portion of an intact antibody, generally including theantigen binding or variable region of the intact antibody or the Fregion of an antibody which retains or has modified FcR bindingcapability. Examples of antibody fragments include linear antibody,single-chain antibody molecules and multispecific antibodies formed fromantibody fragments.

The term “diabodies” refers to small antibody fragments prepared byconstructing sFv fragments (see preceding paragraph) with short linkers(about 5-10) residues) between the V_(H) and V_(L) domains such thatinter-chain but not intra-chain pairing of the V domains is achieved,thereby resulting in a bivalent fragment, i.e., a fragment having twoantigen-binding sites. Bispecific diabodies are heterodimers of two“crossover” sFv fragments in which the V_(H) and V_(L) domains of thetwo antibodies are present on different polypeptide chains. Diabodiesare described in greater detail in, for example, EP 404,097; WO93/11161; Hollinger et al., Proc. Nat'l Acad. Sci. USA 90:6444-48(1993).

As used herein, a “chimeric antibody” refers to an antibody(immunoglobulin), such as an anti-C1s, anti-C1q, anti-C1r, and/oranti-C1 complex antibody of the present disclosure, in which a portionof the heavy and/or light chain is identical with or homologous tocorresponding sequences in antibodies derived from a particular speciesor belonging to a particular antibody class or subclass, while theremainder of the chain(s) is(are) identical with or homologous tocorresponding sequences in antibodies derived from another species orbelonging to another antibody class or subclass, as well as fragments ofsuch antibodies, so long as they exhibit the desired biological activity(U.S. Pat. No. 4,816,567; Morrison et al., Proc. Nat'l Acad. Sci. USA,81:6851-55 (1984)). Chimeric antibodies of interest herein includePRIMATIZED® antibodies wherein the antigen-binding region of theantibody is derived from an antibody produced by, e.g., immunizingmacaque monkeys with an antigen of interest. As used herein, “humanizedantibody” is a subset of “chimeric antibodies.”

“Humanized” forms of non-human (e.g., murine) antibodies, such asanti-C1q, anti-C1s, anti-C1r, and/or anti-C1 complex antibodies of thepresent disclosure, are chimeric antibodies that contain minimalsequence derived from non-human immunoglobulin. In one embodiment, ahumanized antibody is a human immunoglobulin (recipient antibody) inwhich residues from an HVR of the recipient are replaced by residuesfrom an HVR of a non-human species (donor antibody) such as mouse, rat,rabbit or non-human primate having the desired specificity, affinity,and/or capacity. In some instances, FR residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Furthermore, humanized antibodies may comprise residues that are notfound in the recipient antibody or in the donor antibody. Thesemodifications may be made to further refine antibody performance, suchas binding affinity. In general, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin sequence, and all orsubstantially all of the FR regions are those of a human immunoglobulinsequence, although the FR regions may include one or more individual FRresidue substitutions that improve antibody performance, such as bindingaffinity, isomerization, immunogenicity, and the like. The number ofthese amino acid substitutions in the FR is typically no more than 6 inthe H chain, and in the L chain, no more than 3. The humanized antibodyoptionally will also comprise at least a portion of an immunoglobulinconstant region (Fc), typically that of a human immunoglobulin. Forfurther details, see, e.g., Jones et al., Nature 321:522-525 (1986);Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op.Struct. Biol. 2:593-596 (1992). See also, for example, Vaswani andHamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998); Harris,Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross, Curr.Op. Biotech. 5:428-433 (1994); and U.S. Pat. Nos. 6,982,321 and7,087,409.

A “human antibody” is one that possesses an amino-acid sequencecorresponding to that of an antibody, such as an anti-C1q, anti-C1s,anti-C1r, and/or anti-C1 complex antibody of the present disclosure,produced by a human and/or has been made using any of the techniques formaking human antibodies as disclosed herein. This definition of a humanantibody specifically excludes a humanized antibody comprising non-humanantigen-binding residues. Human antibodies can be produced using varioustechniques known in the art, including phage-display libraries.Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J.Mol. Biol., 222:581 (1991). Also available for the preparation of humanmonoclonal antibodies are methods described in Cole et al., MonoclonalAntibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner etal., J. Immunol., 147(1):86-95 (1991). See also van Dijk and van deWinkel, Curr. Opin. Pharmacol. 5:368-74 (2001). Human antibodies can beprepared by administering the antigen to a transgenic animal that hasbeen modified to produce such antibodies in response to antigenicchallenge, but whose endogenous loci have been disabled, e.g., immunizedxenomice (see, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 regardingXENOMOUSE™ technology). See also, for example, Li et al., Proc. Nat'lAcad. Sci. USA, 103:3557-3562 (2006) regarding human antibodiesgenerated via a human B-cell hybridoma technology.

The term “hypervariable region,” “HVR,” or “HV,” when used herein refersto the regions of an antibody-variable domain, such as that of ananti-C1q, anti-C1s, anti-C1r, and/or anti-C1 complex antibody of thepresent disclosure, that are hypervariable in sequence and/or formstructurally defined loops. Generally, antibodies comprise six HVRs;three in the VH (H1, H2, H3), and three in the VL (L1, L2, L3). Innative antibodies, H3 and L3 display the most diversity of the six HVRs,and H3 in particular is believed to play a unique role in conferringfine specificity to antibodies. See, e.g., Xu et al., Immunity 13:37-45(2000); Johnson and Wu in Methods in Molecular Biology 248:1-25 (Lo,ed., Human Press, Totowa, N.J., 2003)). Indeed, naturally occurringcamelid antibodies consisting of a heavy chain only are functional andstable in the absence of light chain. See, e.g., Hamers-Casterman etal., Nature 363:446-448 (1993) and Sheriff et al., Nature Struct. Biol.3:733-736 (1996).

A number of HVR delineations are in use and are encompassed herein. TheHVRs that are Kabat complementarity-determining regions (CDRs) are basedon sequence variability and are the most commonly used (Kabat et al.,supra). Chothia refers instead to the location of the structural loops(Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). The AbM HVRsrepresent a compromise between the Kabat CDRs and Chothia structuralloops, and are used by Oxford Molecular's AbM antibody-modelingsoftware. The “contact” HVRs are based on an analysis of the availablecomplex crystal structures. The residues from each of these HVRs arenoted below.

Loop Kabat AbM Chothia Contact L1 L24-L34 L24-L34 L26-L32 L30-L36 L2L50-L56 L50-L56 L50-L52 L46-L55 L3 L89-L97 L89-L97 L91-L96 L89-L96 H1H31-H35B H26-H35B H26-H32 H30-H35B (Kabat numbering) H1 H31-H35 H26-H35H26-H32 H30-H35 (Chothia numbering) H2 H50-H65 H50-H58 H53-H55 H47-H58H3 H95-H102 H95-H102 H96-H101 H93-H101

HVRs may comprise “extended HVRs” as follows: 24-36 or 24-34 (L1), 46-56or 50-56 (L2), and 89-97 or 89-96 (L3) in the VL, and 26-35 (H1), 50-65or 49-65 (a preferred embodiment) (H2), and 93-102, 94-102, or 95-102(H3) in the VH. The variable-domain residues are numbered according toKabat et al., supra, for each of these extended-HVR definitions.

“Framework” or “FR” residues are those variable-domain residues otherthan the HVR residues as herein defined.

The phrase “variable-domain residue-numbering as in Kabat” or“amino-acid-position numbering as in Kabat,” and variations thereof,refers to the numbering system used for heavy-chain variable domains orlight-chain variable domains of the compilation of antibodies in Kabatet al., supra. Using this numbering system, the actual linear amino acidsequence may contain fewer or additional amino acids corresponding to ashortening of, or insertion into, a FR or HVR of the variable domain.For example, a heavy-chain variable domain may include a single aminoacid insert (residue 52a according to Kabat) after residue 52 of H2 andinserted residues (e.g., residues 82a, 82b, and 82c, etc. according toKabat) after heavy-chain FR residue 82. The Kabat numbering of residuesmay be determined for a given antibody by alignment at regions ofhomology of the sequence of the antibody with a “standard” Kabatnumbered sequence.

The Kabat numbering system is generally used when referring to a residuein the variable domain (approximately residues 1-107 of the light chainand residues 1-113 of the heavy chain) (e.g., Kabat et al., Sequences ofImmunological Interest. 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991)). The “EU numbering system”or “EU index” is generally used when referring to a residue in animmunoglobulin heavy chain constant region (e.g., the EU index reportedin Kabat et al., supra). The “EU index as in Kabat” refers to theresidue numbering of the human IgG1 EU antibody. Unless stated otherwiseherein, references to residue numbers in the variable domain ofantibodies means residue numbering by the Kabat numbering system. Unlessstated otherwise herein, references to residue numbers in the constantdomain of antibodies means residue numbering by the EU numbering system(e.g., see United States Patent Publication No. 2010-280227).

An “acceptor human framework” as used herein is a framework comprisingthe amino acid sequence of a VL or VH framework derived from a humanimmunoglobulin framework or a human consensus framework. An acceptorhuman framework “derived from” a human immunoglobulin framework or ahuman consensus framework may comprise the same amino acid sequencethereof, or it may contain pre-existing amino acid sequence changes. Insome embodiments, the number of pre-existing amino acid changes are 10or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 orless, 3 or less, or 2 or less. Where pre-existing amino acid changes arepresent in a VH, preferable those changes occur at only three, two, orone of positions 71H, 73H and 78H; for instance, the amino acid residuesat those positions may by 71A, 73T and/or 78A. In one embodiment, the VLacceptor human framework is identical in sequence to the VL humanimmunoglobulin framework sequence or human consensus framework sequence.

A “human consensus framework” is a framework that represents the mostcommonly occurring amino acid residues in a selection of humanimmunoglobulin VL or VH framework sequences. Generally, the selection ofhuman immunoglobulin VL or VH sequences is from a subgroup of variabledomain sequences. Generally, the subgroup of sequences is a subgroup asin Kabat et al., Sequences of Proteins of Immunological Interest, 5thEd. Public Health Service, National Institutes of Health, Bethesda, Md.(1991). Examples include for the VL, the subgroup may be subgroup kappaI, kappa II, kappa III or kappa IV as in Kabat et al., supra.Additionally, for the VH, the subgroup may be subgroup I, subgroup II,or subgroup III as in Kabat et al., supra.

An “amino-acid modification” at a specified position, e.g., of ananti-C1q, anti-C1s, anti-C1r, and/or anti-C1 complex antibody of thepresent disclosure, refers to the substitution or deletion of thespecified residue, or the insertion of at least one amino acid residueadjacent the specified residue. Insertion “adjacent” to a specifiedresidue means insertion within one to two residues thereof. Theinsertion may be N-terminal or C-terminal to the specified residue. Thepreferred amino acid modification herein is a substitution.

An “affinity-matured” antibody, such as an anti-C1q, anti-C1s, anti-C1r,and/or anti-C1 complex antibody of the present disclosure, is one withone or more alterations in one or more HVRs thereof that result in animprovement in the affinity of the antibody for antigen, compared to aparent antibody that does not possess those alteration(s). In oneembodiment, an affinity-matured antibody has nanomolar or even picomolaraffinities for the target antigen. Affinity-matured antibodies areproduced by procedures known in the art. For example, Marks et al.,Bio/Technology 10:779-783 (1992) describes affinity maturation by VH-and VL-domain shuffling. Random mutagenesis of HVR and/or frameworkresidues is described by, for example: Barbas et al. Proc Nat. Acad.Sci. USA 91:3809-3813 (1994); Schier et al. Gene 169:147-155 (1995);Yelton et al. J. Immunol. 155:1994-2004 (1995); Jackson et al., J.Immunol. 154(7):3310-9 (1995); and Hawkins et al, J. Mol. Biol.226:889-896 (1992).

As use herein, the term “specifically recognizes” or “specificallybinds” refers to measurable and reproducible interactions such asattraction or binding between a target and an antibody, such as ananti-C1q, anti-C1s, anti-C1r, and/or anti-C1 complex antibody of thepresent disclosure, that is determinative of the presence of the targetin the presence of a heterogeneous population of molecules includingbiological molecules. For example, an antibody, such as an anti-C1q,anti-C1s, anti-C1r, and/or anti-C1 complex antibody of the presentdisclosure, that specifically or preferentially binds to a target or anepitope is an antibody that binds this target or epitope with greateraffinity, avidity, more readily, and/or with greater duration than itbinds to other targets or other epitopes of the target. It is alsounderstood by reading this definition that, for example, an antibody (ora moiety) that specifically or preferentially binds to a first targetmay or may not specifically or preferentially bind to a second target.As such, “specific binding” or “preferential binding” does notnecessarily require (although it can include) exclusive binding. Anantibody that specifically binds to a target may have an associationconstant of at least about 10³ M⁻¹ or 10⁴M⁻¹, sometimes about 10⁵M⁻¹ or10⁶M⁻¹, in other instances about 10⁶ M⁻¹ or 10⁷ M⁻¹, about 10⁸M⁻¹ to 10⁹M⁻¹, or about 10¹⁰ M⁻¹ to 10¹¹ M⁻¹ or higher. A variety of immunoassayformats can be used to select antibodies specifically immunoreactivewith a particular protein. For example, solid-phase ELISA immunoassaysare routinely used to select monoclonal antibodies specificallyimmunoreactive with a protein. See, e.g., Harlow and Lane (1988)Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, NewYork, for a description of immunoassay formats and conditions that canbe used to determine specific immunoreactivity.

As used herein, an “interaction” between a complement protein, such ascomplement factors C1q, C1s, and C1r and a second protein encompasses,without limitation, protein-protein interaction, a physical interaction,a chemical interaction, binding, covalent binding, and ionic binding. Asused herein, an antibody “inhibits interaction” between two proteinswhen the antibody disrupts, reduces, or completely eliminates aninteraction between the two proteins. An antibody of the presentdisclosure, or fragment thereof, “inhibits interaction” between twoproteins when the antibody or fragment thereof binds to one of the twoproteins.

A “blocking” antibody, an “antagonist” antibody, an “inhibitory”antibody, or a “neutralizing” antibody is an antibody, such as ananti-C1q, anti-C1s, anti-C1r, and/or anti-C1 complex antibody of thepresent disclosure that inhibits or reduces one or more biologicalactivities of the antigen it binds, such as interactions with one ormore proteins. In some embodiments, blocking antibodies, antagonistantibodies, inhibitory antibodies, or “neutralizing” antibodiessubstantially or completely inhibit one or more biological activities orinteractions of the antigen.

Antibody “effector functions” refer to those biological activitiesattributable to the Fc region (a native sequence Fc region or amino acidsequence variant Fc region) of an antibody, and vary with the antibodyisotype.

The term “Fc region” herein is used to define a C-terminal region of animmunoglobulin heavy chain, including native-sequence Fc regions andvariant Fc regions. Although the boundaries of the Fc region of animmunoglobulin heavy chain might vary, the human IgG heavy-chain Fcregion is usually defined to stretch from an amino acid residue atposition Cys226, or from Pro230, to the carboxyl-terminus thereof. TheC-terminal lysine (residue 447 according to the EU numbering system) ofthe Fc region may be removed, for example, during production orpurification of the antibody, or by recombinantly engineering thenucleic acid encoding a heavy chain of the antibody. Accordingly, acomposition of intact antibodies may comprise antibody populations withall K447 residues removed, antibody populations with no K447 residuesremoved, and antibody populations having a mixture of antibodies withand without the K447 residue. Suitable native-sequence Fc regions foruse in the antibodies of the invention include human IgG1, IgG2, IgG3and IgG4.

A “native sequence Fc region” comprises an amino acid sequence identicalto the amino acid sequence of an Fc region found in nature. Nativesequence human Fc regions include a native sequence human IgG1 Fc region(non-A and A allotypes); native sequence human IgG2 Fc region; nativesequence human IgG3 Fc region; and native sequence human IgG4 Fc regionas well as naturally occurring variants thereof.

A “variant Fc region” comprises an amino acid sequence which differsfrom that of a native sequence Fc region by virtue of at least one aminoacid modification, preferably one or more amino acid substitution(s).Preferably, the variant Fc region has at least one amino acidsubstitution compared to a native sequence Fc region or to the Fc regionof a parent polypeptide, e.g. from about one to about ten amino acidsubstitutions, and preferably from about one to about five amino acidsubstitutions in a native sequence Fc region or in the Fc region of theparent polypeptide. The variant Fc region herein will preferably possessat least about 80% homology with a native sequence Fc region and/or withan Fc region of a parent polypeptide, and most preferably at least about90% homology therewith, more preferably at least about 95% homologytherewith.

“Fc receptor” or “FcR” describes a receptor that binds to the Fc regionof an antibody. The preferred FcR is a native sequence human FcR.Moreover, a preferred FcR is one which binds an IgG antibody (a gammareceptor) and includes receptors of the FcγRI, FcγRII, and FcγRIIIsubclasses, including allelic variants and alternatively spliced formsof these receptors, FcγRII receptors include FcγRIIA (an “activatingreceptor”) and FcγRIIB (an “inhibiting receptor”), which have similaramino acid sequences that differ primarily in the cytoplasmic domainsthereof. Activating receptor FcγRIIA contains an immunoreceptortyrosine-based activation motif (“ITAM”) in its cytoplasmic domain.Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-basedinhibition motif (“ITIM”) in its cytoplasmic domain. (see, e.g., M.Daëron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed inRavetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991); Capel et al.,Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med.126: 330-41 (1995). Other FcRs, including those to be identified in thefuture, are encompassed by the term “FcR” herein. FcRs can also increasethe serum half-life of antibodies.

Binding to FcRn in vivo and serum half-life of human FcRn high-affinitybinding polypeptides can be assayed, e.g., in transgenic mice ortransfected human cell lines expressing human FcRn, or in primates towhich the polypeptides having a variant Fc region are administered. WO2004/42072 (Presta) describes antibody variants with improved ordiminished binding to FcRs. See also, e.g., Shields et al., J. Biol.Chem. 9(2):6591-6604 (2001).

The term “k_(on)”, as used herein, is intended to refer to the rateconstant for association of an antibody to an antigen.

The term “k_(off)”, as used herein, is intended to refer to the rateconstant for dissociation of an antibody from the antibody/antigencomplex.

The term “K_(D)”, as used herein, is intended to refer to theequilibrium dissociation constant of an antibody-antigen interaction.

As used herein, “percent (%) amino acid sequence identity” and“homology” with respect to a peptide, polypeptide or antibody sequencerefers to the percentage of amino acid residues in a candidate sequencethat are identical with the amino acid residues in the specific peptideor polypeptide sequence, after aligning the sequences and introducinggaps, if necessary, to achieve the maximum percent sequence identity,and not considering any conservative substitutions as part of thesequence identity. Alignment for purposes of determining percent aminoacid sequence identity can be achieved in various ways that are withinthe skill in the art, for instance, using publicly available computersoftware such as BLAST, BLAST-2, ALIGN or MEGALIGN™ (DNASTAR) software.Those skilled in the art can determine appropriate parameters formeasuring alignment, including any algorithms known in the art needed toachieve maximal alignment over the full length of the sequences beingcompared.

An “isolated” molecule or cell is a molecule or a cell that isidentified and separated from at least one contaminant molecule or cellwith which it is ordinarily associated in the environment in which itwas produced. Preferably, the isolated molecule or cell is free ofassociation with all components associated with the productionenvironment. The isolated molecule or cell is in a form other than inthe form or setting in which it is found in nature. Isolated moleculestherefore are distinguished from molecules existing naturally in cells;isolated cells are distinguished from cells existing naturally intissues, organs, or individuals. In some embodiments, the isolatedmolecule is an anti-C1q, anti-C1s, anti-C1r, and/or anti-C1 complexantibody of the present disclosure. In other embodiments, the isolatedcell is a host cell or hybridoma cell producing an anti-C1q, anti-C1s,anti-C1r, and/or anti-C1 complex antibody of the present disclosure.

An “isolated” nucleic acid molecule encoding an antibody, such as ananti-C1q, anti-C1s, anti-C1r, and/or anti-C1 complex antibody of thepresent disclosure, is a nucleic acid molecule that is identified andseparated from at least one contaminant nucleic acid molecule with whichit is ordinarily associated in the environment in which it was produced.Preferably, the isolated nucleic acid is free of association with allcomponents associated with the production environment. The isolatednucleic acid molecules encoding the polypeptides and antibodies hereinis in a form other than in the form or setting in which it is found innature. Isolated nucleic acid molecules therefore are distinguished fromnucleic acid encoding the polypeptides and antibodies herein existingnaturally in cells.

The term “vector,” as used herein, is intended to refer to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid,” which refers to acircular double stranded DNA into which additional DNA segments may beligated. Another type of vector is a phage vector. Another type ofvector is a viral vector, wherein additional DNA segments may be ligatedinto the viral genome. Certain vectors are capable of autonomousreplication in a host cell into which they are introduced (e.g.,bacterial vectors having a bacterial origin of replication and episomalmammalian vectors). Other vectors (e.g., non-episomal mammalian vectors)can be integrated into the genome of a host cell upon introduction intothe host cell, and thereby are replicated along with the host genome.Moreover, certain vectors are capable of directing the expression ofgenes to which they are operatively linked. Such vectors are referred toherein as “recombinant expression vectors,” or simply, “expressionvectors.” In general, expression vectors of utility in recombinant DNAtechniques are often in the form of plasmids. In the presentspecification, “plasmid” and “vector” may be used interchangeably as theplasmid is the most commonly used form of vector.

“Polynucleotide,” or “nucleic acid,” as used interchangeably herein,refer to polymers of nucleotides of any length, and include DNA and RNA.The nucleotides can be deoxyribonucleotides, ribonucleotides, modifiednucleotides or bases, and/or their analogs, or any substrate that can beincorporated into a polymer by DNA or RNA polymerase or by a syntheticreaction. A polynucleotide may comprise modified nucleotides, such asmethylated nucleotides and their analogs. If present, modification tothe nucleotide structure may be imparted before or after assembly of thepolymer. The sequence of nucleotides may be interrupted bynon-nucleotide components. A polynucleotide may comprise modification(s)made after synthesis, such as conjugation to a label. Other types ofmodifications include, for example, “caps,” substitution of one or moreof the naturally occurring nucleotides with an analog, internucleotidemodifications such as, for example, those with uncharged linkages (e.g.,methyl phosphonates, phosphotriesters, phosphoamidates, carbamates,etc.) and with charged linkages (e.g., phosphorothioates,phosphorodithioates, etc.), those containing pendant moieties, such as,for example, proteins (e.g., nucleases, toxins, antibodies, signalpeptides, ply-L-lysine, etc.), those with intercalators (e.g., acridine,psoralen, etc.), those containing chelators (e.g., metals, radioactivemetals, boron, oxidative metals, etc.), those containing alkylators,those with modified linkages (e.g., alpha anomeric nucleic acids, etc.),as well as unmodified forms of the polynucleotides(s). Further, any ofthe hydroxyl groups ordinarily present in the sugars may be replaced,for example, by phosphonate groups, phosphate groups, protected bystandard protecting groups, or activated to prepare additional linkagesto additional nucleotides, or may be conjugated to solid or semi-solidsupports. The 5′ and 3′ terminal OH can be phosphorylated or substitutedwith amines or organic capping group moieties of from 1 to 20 carbonatoms. Other hydroxyls may also be derivatized to standard protectinggroups. Polynucleotides can also contain analogous forms of ribose ordeoxyribose sugars that are generally known in the art, including, forexample, 2′-O-methyl-, 2′-O-allyl-, 2′-fluoro- or 2′-azido-ribose,carbocyclic sugar analogs, α-anomeric sugars, epimeric sugars such asarabinose, xyloses or lyxoses, pyranose sugars, furanose sugars,sedoheptuloses, acyclic analogs, and basic nucleoside analogs such asmethyl riboside. One or more phosphodiester linkages may be replaced byalternative linking groups. These alternative linking groups include,but are not limited to, embodiments wherein phosphate is replaced byP(O)S (“thioate”), P(S)S (“dithioate”), (O)NR2 (“amidate”), P(O)R,P(O)OR′, CO, or CH2 (“formacetal”), in which each R or R′ isindependently H or substituted or unsubstituted alkyl (1-20 C)optionally containing an ether (—O—) linkage, aryl, alkenyl, cycloalkyl,cycloalkenyl or araldyl. Not all linkages in a polynucleotide need beidentical. The preceding description applies to all polynucleotidesreferred to herein, including RNA and DNA.

A “host cell” includes an individual cell or cell culture that can be orhas been a recipient for vector(s) for incorporation of polynucleotideinserts. Host cells include progeny of a single host cell, and theprogeny may not necessarily be completely identical (in morphology or ingenomic DNA complement) to the original parent cell due to natural,accidental, or deliberate mutation. A host cell includes cellstransfected in vivo with a polynucleotide(s) of this invention.

“Carriers” as used herein include pharmaceutically acceptable carriers,excipients, or stabilizers that are nontoxic to the cell or mammal beingexposed thereto at the dosages and concentrations employed. Often thephysiologically acceptable carrier is an aqueous pH buffered solution.Examples of physiologically acceptable carriers include buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid; low molecular weight (less than about 10 residues)polypeptide; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, arginine or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugaralcohols such as mannitol or sorbitol; salt-forming counterions such assodium; and/or nonionic surfactants such as TWEEN™, polyethylene glycol(PEG), and PLURONICS™.

The term “about” as used herein refers to the usual error range for therespective value readily known to the skilled person in this technicalfield. Reference to “about” a value or parameter herein includes (anddescribes) embodiments that are directed to that value or parameter perse.

As used herein and in the appended claims, the singular forms “a,” “an,”and “the” include plural reference unless the context clearly indicatesotherwise. For example, reference to an “antibody” is a reference tofrom one to many antibodies, such as molar amounts, and includesequivalents thereof known to those skilled in the art, and so forth.

It is understood that aspect and embodiments of the invention describedherein include “comprising,” “consisting,” and “consisting essentiallyof” aspects and embodiments.

Overview

In certain aspects, the present disclosure provides methods of treating,preventing, or reducing risk of Guillain-Barre Syndrome (GBS). Withoutwishing to be bound by theory, it is believed that inhibition of theclassical pathway of complement activation is an effective therapeuticstrategy for the treatment of GBS (FIG. 5A). It is further believed thateffective strategies for inhibiting the classical pathway includeinhibiting the interaction between C1q and autoantibodies (e.g.,anti-ganglioside autoantibodies), i inhibiting the interaction betweenC1q and C1r or C1s, blocking the catalytic activity of C1r or C1s, andblocking the interactions between C1r or C1s and their respectivesubstrates (FIG. 5A). It is also believed that effective agents for theinhibition of the classical complement pathway include neutralizingantibodies for C1q, C1s, C1r, and/or C1 complex (FIG. 5B).

Accordingly, certain aspects of the present disclosure relates toanti-C1q antibodies, anti-C1s antibodies, anti-C1r antibodies, and/oranti-C1 complex antibodies for use in treating, preventing, or reducingrisk of Guillain-Barre Syndrome (GBS) in individuals in need thereof.

In one aspect, the present disclosure provides methods for treating orpreventing GBS an individual by administering to the individual atherapeutically effective amount of at least one antibody, wherein theat least one antibody is an anti-C1q antibody. In some embodiments, theanti-C1q antibody is a C1q neutralizing antibody. In some embodiments,the anti-C1q antibody binds to C1 complex. In some embodiments, theanti-C1q antibody inhibits the interaction between C1q and anautoantibody, between C1q and C1r, and/or between C1q and C1s. In someembodiments, the individual has GBS. In certain preferred embodiments,the individual is a human.

In another aspect, the present disclosure provides methods for treatingor preventing GBS an individual by administering to the individual atherapeutically effective amount of at least one antibody, wherein theat least one antibody is an anti-C1s antibody. In some embodiments, theanti-C1s antibody is a C1s neutralizing antibody. In some embodiments,the anti-C1s antibody binds to C1 complex. In some embodiments, theanti-C1s antibody inhibits the interaction between C1s and C1q, betweenC1s and C1r, and/or between C1s and its substrates C2 and C4. In someembodiments, the anti-C1s antibody inhibits the catalytic activity ofC1s or the processing of pro-C1s into an active protease.

In another aspect, the present disclosure provides methods for treatingor preventing GBS an individual by administering to the individual atherapeutically effective amount of at least one antibody, wherein theat least one antibody is an anti-C1r antibody. In some embodiments, theanti-C1r antibody is a C1r neutralizing antibody. In some embodiments,the anti-C1r antibody binds to C1 complex. In some embodiments, theanti-C1r antibody inhibits the interaction between C1r and C1q and/orbetween C1r and C1s. In some embodiments, the anti-C1r antibody inhibitsthe catalytic activity of C1r or the processing of pro-C1r to an activeprotease.

In another aspect, the present disclosure provides methods for treatingor preventing GBS an individual by administering to the individual atherapeutically effective amount of at least one antibody, wherein theat least one antibody is an anti-C1 complex antibody. In someembodiments, the anti-C1 complex antibody is a C1 complex neutralizingantibody. In some embodiments, the anti-C1 complex antibody binds toC1q, C1s, and/or C1r. In some embodiments, the anti-C1 complex antibodyinhibits C1r activation and/or C1s activation. In some embodiments, theanti-C1 complex antibody prevents the ability of C1r to act on C2 or C4and/or the ability of C1s to act on C2 or C4. In some embodiments, theanti-C1 complex antibody binds to a combinatorial epitope within the C1complex, wherein said combinatorial epitope is comprised of C1q and C1s;C1q and C1r; C1r and C1s; or C1q, C1r, and C1s.

Further aspects of the present disclosure provide neutralizing anti-C1santibodies and uses therefore. In some embodiments, the presentdisclosure provides neutralizing monoclonal murine anti-C1s antibodies5A1 and 5C12, which are produced by hybridoma cell lines deposited withATCC on May 15, 2013 and having ATCC Accession Numbers PTA-120351 andPTA-120352, and antibodies derived from anti-C1s antibodies 5A1 and5C12. Uses for neutralizing anti-C1s antibodies include, withoutlimitation, the detection of complement factor C1s. Additionalnon-limiting uses include the inhibition of the classical pathway ofcomplement activation, e.g., in cases where the classical complementpathway is activated by autoantibodies, such as anti-gangliosideautoantibodies. Further non-limiting uses for neutralizing anti-C1santibodies include the diagnosis and treatment of disorders associatedwith increased activation of the classical complement pathway, inparticular autoimmune disorders, such as GBS, and neurodegenerativedisorders, including neurodegenerative disorders associated with synapseloss.

In a further aspect, the present disclosure provides an anti-C1smonoclonal antibody which binds to and neutralizes a biological activityof C1s. In some embodiments, the anti-C1s antibodies of this disclosurealso bind to the C1s proenzyme. The neutralizing anti-C1s antibodies mayneutralize, without limitation, one or more biological activities ofC1s. Such biological activities include, without limitation, C1s bindingto C1q or C1s binding to C1r, as well as C1s binding to C2 or C4. Othernon-limiting biological activities of C1s include the proteolytic enzymeactivity of C1s or the conversion of the C1s proenzyme (C1s-pro) to anactive C1s protease. Other biological activities include the cleavage ofC4. Other non-limiting biological activities of C1s include theactivation of the classical complement activation pathway, theactivation of antibody and complement dependent cytotoxicity, and C1Fhemolysis. In some embodiments, the anti-C1s antibodies of thisdisclosure may bind to C1 complex.

In another aspect, the present disclosure provides an isolated nucleicacid molecule encoding an antibody of this disclosure.

The present disclosure also provides isolated host cells containing anucleic acid molecule that encodes an antibody of this disclosure. Insome embodiments, isolated host cell lines are provided that can producethe neutralizing monoclonal murine antibodies 5A1 and 5C12. Suchisolated host cell lines were deposited with ATCC on May 15, 2013 andhave ATCC Accession Numbers PTA-120351 and PTA-120352. Additionally,pharmaceutical compositions are provided containing C1s neutralizingantibodies of this disclosure in combination with pharmaceuticallyacceptable carriers.

The present disclosure further provides methods of using the C1sneutralizing antibodies of this disclosure to treat or prevent anautoimmune or neurodegenerative disease in a subject in need of suchtreatment, to detect synapses in an individual having an autoimmune orneurodegenerative disease, and to detect synapses in a biologicalsample. The present disclosure also provides diagnostic kits containingthe C1s neutralizing antibodies of this disclosure.

Complement Proteins

The methods of this disclosure involve administering or using antibodiesthat specifically recognize complement factors C1q, C1s, C1r, and/or theC1 complex of the classical complement activation pathway. Certainaspects of the present disclosure further involve antibodies thatspecifically recognize complement factors C1q and/or C1q in the C1complex, and C1s and/or C1s in the C1 complex of the classicalcomplement activation pathway. The recognized complement factors may bederived, without limitation, from any organism having a complementsystem, including any mammalian organism such as human, mouse, rat,rabbit, monkey, dog, cat, cow, horse, camel, sheep, goat, or pig.

As used herein “C1 complex” refers to a protein complex that mayinclude, without limitation, one C1q protein, two C1r proteins, and twoC1s proteins (e.g., C1qr²s²).

As used herein “complement factors C1q, C1s, or C1r” refers to both wildtype sequences and naturally occurring variant sequences.

C1q

A non-limiting example of a complement factor C1q recognized byantibodies of this invention is human C1q, including the threepolypeptide chains A, B, and C:

C1q, chain A (homo sapiens), Accession No. Protein Data Base:NP_057075.1; GenBank No.: NM_015991:

<gi|7705753|ref|NP_057075.1| complement C1qsubcomponent subunit A precursor [Homo sapiens] (SEQ ID NO: 1)MEGPRGWLVLCVLAISLASMVTEDLCRAPDGKKGEAGRPGRRGRPGLKGEQGEPGAPGIRTGIQGLKGDQGEPGPSGNPGKVGYPGPSGPLGARGIPGIKGTKGSPGNIKDQPRPAFSAIRRNPPMGGNVVIFDTVITNQEEPYQNHSGRFVCTVPGYYYFTFQVLSQWEICLSIVSSSRGQVRRSLGFCDTTNKGLFQVVSGGMVLQLQQGDQVWVEKDPKKGHIYQGSEADSVFSGFLIFPSA C1q, chain B (homo sapiens), Accession No. Protein Data Base:NP_000482.3; GenBank No.: NM_000491.3:

>gi|87298828|ref|NP_000482.3| complement C1qsubcomponent subunit B precursor [Homo sapiens] (SEQ ID NO: 2)MMMKIPWGSIPVLMLLLLLGLIDISQAQLSCTGPPAIPGIPGIPGTPGPDGQPGTPGIKGEKGLPGLAGDHGEFGEKGDPGIPGNPGKVGPKGPMGPKGGPGAPGAPGPKGESGDYKATQKIAFSATRTINVPLRRDQTIRFDHVITNMNNNYEPRSGKFTCKVPGLYYFTYHASSRGNLCVNLMRGRERAQKVVTFCDYAYNTFQVTTGGMVLKLEQGENVFLQATDKNSLLGMEGANSIFSGFLLFPD MEA C1q, chain C (homo sapiens), Accession No. Protein Data Base:NP_001107573.1; GenBank No.: NM_001114101.1:

>gi|166235903|ref|NP_001107573.1| complement C1qsubcomponent subunit C precursor [Homo sapiens] (SEQ ID NO: 3)MDVGPSSLPHLGLKLLLLLLLLPLRGQANTGCYGIPGMPGLPGAPGKDGYDGLPGPKGEPGIPAIPGIRGPKGQKGEPGLPGHPGKNGPMGPPGMPGVPGPMGIPGEPGEEGRYKQKFQSVFTVTRQTHQPPAPNSLIRFNAVLTNPQGDYDTSTGKFTCKVPGLYYFVYHASHTANLCVLLYRSGVKVVTFCGHTSKTNQVNSGGVLLRLQVGEEVWLAVNDYYDMVGIQGSDSVFSGFLLFPD 

Accordingly, an anti-C1q antibody of the present disclosure may bind topolypeptide chain A, polypeptide chain B, and/or polypeptide chain C ofa C1q protein. In some embodiments, an anti-C1q antibody of the presentdisclosure binds to polypeptide chain A, polypeptide chain B, and/orpolypeptide chain C of human C1q or a homolog thereof, such as mouse,rat, rabbit, monkey, dog, cat, cow, horse, camel, sheep, goat, or pigC1q.

C1s

A non-limiting example of a complement factor C1s recognized byantibodies of this invention is human C1s (Accession No. Protein DataBase: NP_958850.1; GenBank No.: NM_201442.2):

>gi|41393602|ref|NP_958850.1| complement C1ssubcomponent precursor [Homo sapiens] (SEQ ID NO: 4)MWCIVLFSLLAWVYAEPTMYGEILSPNYPQAYPSEVEKSWDIEVPEGYGIHLYFTHLDIELSENCAYDSVQIISGDTEEGRLCGQRSSNNPHSPIVEEFQVPYNKLQVIFKSDFSNEERFTGFAAYYVATDINECTDFVDVPCSHFCNNFIGGYFCSCPPEYFLHDDMKNCGVNCSGDVFTALIGEIASPNYPKPYPENSRCEYQIRLEKGFQVVVTLRREDFDVEAADSAGNCLDSLVFVAGDRQFGPYCGHGFPGPLNIETKSNALDIIFQTDLTGQKKGWKLRYHGDPMPCPKEDTPNSVWEPAKAKYVFRDVVQITCLDGFEVVEGRVGATSFYSTCQSNGKWSNSKLKCQPVDCGIPESIENGKVEDPESTLFGSVIRYTCEEPYYYMENGGGGEYHCAGNGSWVNEVLGPELPKCVPVCGVPREPFEEKQRIIGGSDADIKNFPWQVFFDNPWAGGALINEYWVLTAAHVVEGNREPTMYVGSTSVQTSRLAKSKMLTPEHVFIHPGWKLLEVPEGRTNFDNDIALVRLKDPVKMGPTVSPICLPGTSSDYNLMDGDLGLISGWGRTEKRDRAVRLKAARLPVAPLRKCKEVKVEKPTADAEAYVFTPNMICAGGEKGMDSCKGDSGGAFAVQDPNDKTKFYAAGLVSWGPQCGTYGLYTRVKNYVDWIMKTMQENSTPRED 

Accordingly, an anti-C1s antibody of the present disclosure may bind tohuman C1s or a homolog thereof, such as mouse, rat, rabbit, monkey, dog,cat, cow, horse, camel, sheep, goat, or pig C1s.

C1r

A non-limiting example of a complement factor C1r recognized byantibodies of this invention is human C1r (Accession No. Protein DataBase: NP_001724.3; GenBank No.: NM_001733.4):

>gi|66347875|ref|NP_001724.3| complement C1rsubcomponent precursor [Homo sapiens] (SEQ ID NO: 5)MWLLYLLVPALFCRAGGSIPIPQKLFGEVTSPLFPKPYPNNFETTTVITVPTGYRVKLVFQQDLEPSEGCFYDYVKISADKKSLGRFCGQLGSPLGNPPGKKEFMSQGNKMLLTFHTDFSNEENGTIMFYKGFLAYYQAVDLDECASRSKLGEEDPQPQCQHLCHNYVGGYFCSCRPGYELQEDRHSCQAECSSELYTEASGYISSLEYPRSYPPDLRCNYSIRVERGLTLHLKFLEPFDIDDHQQVHCPYDQLQIYANGKNIGEFCGKQRPPDLDTSSNAVDLLFFTDESGDSRGWKLRYTTEIIKCPQPKTLDEFTIIQNLQPQYQFRDYFIATCKQGYQLIEGNQVLHSFTAVCQDDGTWHRAMPRCKIKDCGQPRNLPNGDFRYTTTMGVNTYKARIQYYCHEPYYKMQTRAGSRESEQGVYTCTAQGIWKNEQKGEKIPRCLPVCGKPVNPVEQRQRIIGGQKAKMGNFPWQVFTNIHGRGGGALLGDRWILTAAHTLYPKEHEAQSNASLDVFLGHTNVEELMKLGNHPIRRVSVHPDYRQDESYNFEGDIALLELENSVTLGPNLLPICLPDNDTFYDLGLMGYVSGFGVMEEKIAHDLRFVRLPVANPQACENWLRGKNRMDVFSQNMFCAGHPSLKQDACQGDSGGVFAVRDPNTDRWVATGIVSWGIGCSRGYGFYTKVLNYVDWIKKEM EEED 

Accordingly, an anti-C1r antibody of the present disclosure may bind tohuman C1r or a homolog thereof, such as mouse, rat, rabbit, monkey, dog,cat, cow, horse, camel, sheep, goat, or pig C1r.

Anti-C1q, anti-C1s, and anti-C1r Antibodies

The antibodies of the present disclosure recognize complement factorsC1q, C1r, and/or C1s; and/or the C1 complex of the classical complementactivation pathway. In some embodiments, the antibodies neutralize theactivity of complement factors C1q, C1r, and/or C1s. In someembodiments, the antibodies inhibit the interaction between complementfactors C1q, C1r, and/or C1s and autoantibodies, other complementfactors, or complement protease substrates such as C2 and C4. In someembodiments, the antibodies inhibit the catalytic activity of the serineproteases C1s and C1r or inhibit the processing of a serine proteasepro-form to an active protease. In some embodiments the antibodiesinhibit the classical pathway. In certain embodiments the antibodiesfurther inhibit the alternative pathway. In some embodiments, theantibodies inhibit autoantibody- and complement-dependent cytotoxicity(CDC). In some embodiments, the antibodies inhibit complement-dependentcell-mediated cytotoxicity (CDCC).

Anti-C1q Antibodies

The anti-C1q antibodies of this disclosure recognize and bind to C1qand/or C1q in the C1 complex of the classical complement activationpathway. In some embodiments, the anti-C1q antibodies specifically bindto a human C1q. In some embodiments, the anti-C1q antibodiesspecifically bind to a human and a mouse C1q. In some embodiments, theanti-C1q antibodies specifically bind to a human, a mouse, and a ratC1q.

In some embodiments, the anti-C1q antibodies of this disclosureneutralize a biological activity of complement factor C1q. In someembodiments, the antibodies inhibit the interaction between complementfactor C1q and other complement factors (such as C1r or C1s) or betweenC1q and an antibody (such as an autoantibody). In some embodiments, theantibodies inhibit the interaction between complement factor C1q and anon-complement factor. A non-complement factor may includephosphatidylserine, pentraxin-3, C-reactive protein (CRP), globular C1qreceptor (gC1qR), complement receptor 1 (CR1), β-amyloid, andcalreticulin. In some embodiments, the antibodies inhibit the classicalcomplement activation pathway. In certain embodiments, the antibodiesfurther inhibit the alternative pathway. In some embodiments, theantibodies inhibit autoantibody- and complement-dependent cytotoxicity(CDC). In some embodiments, the antibodies inhibit complement-dependentcell-mediated cytotoxicity (CDCC). In some embodiments, the antibodiesinhibit B-cell antibody production, dendritic cell maturation, T-cellproliferation, cytokine production, or microglia activation. In someembodiments, the antibodies inhibit the Arthus reaction. In someembodiments, the antibodies inhibit phagocytosis of synapses or nerveendings. In some embodiments, the antibodies inhibit the activation ofcomplement receptor 3 (CR3/C3) expressing cells.

The functional properties of the anti-C1q antibodies of this invention,such as dissociation constants for antigens, inhibition ofprotein-protein interactions (e.g., C1q-autoantibody interactions),inhibition of autoantibody-dependent and complement-dependentcytotoxicity (CDC), inhibition of complement-dependent cell-mediatedcytotoxicity (CDCC), or lesion formation, may, without limitation, bemeasured in in vitro, ex vivo, or in vivo experiments.

The functional properties of the antibodies of this invention, such asdissociation constants for antigens, inhibition of protein-proteininteractions (e.g., C1q-autoantibody interactions), inhibition ofautoantibody-dependent and complement-dependent cytotoxicity (CDC),inhibition of complement-dependent cell-mediated cytotoxicity (CDCC),and/or lesion formation, may, without limitation, be measured by invitro, ex vivo, or in vivo experiments.

The dissociation constants (K_(D)) of the anti-C1q antibodies for C1qmay be less than 100 nM, less than 90 nM, less than 80 nM, less than 70nM, less than 60 nM, less than 50 nM, less than 40 nM, less than 30 nM,less than 20 nM, less than 10 nM, less than 9 nM, less than 8 nM, lessthan 7 nM, less than 6 nM, less than 5 nM, less than 4 nM, less than 3nM, less than 2 nM, less than 1 nM, less than 0.5 nM, less than 0.1 nM,less than 0.05 nM, less than 0.01 nM, or less than 0.005 nM. In someembodiments, dissociation constants are less than 100 pM. In certainembodiments, the dissociation constants of the anti-C1q antibody areless than 100 pM for human C1q and less than 100 pM for mouse C1q.Antibody dissociation constants for antigens other than C1q may be least5-fold, at least 10-fold, at least 100-fold, at least 1,000-fold, atleast 10,000-fold, or at least 100,000-fold higher that the dissociationconstants for C1q. For example, the dissociation constant of a C1qantibody of this disclosure may be at least 1,000-fold higher for C1sthan for C1q. Dissociation constants may be determined through anyanalytical technique, including any biochemical or biophysical techniquesuch as ELISA, surface plasmon resonance (SPR), bio-layer interferometry(see, e.g., Octet System by ForteBio), isothermal titration calorimetry(ITC), differential scanning calorimetry (DSC), circular dichroism (CD),stopped-flow analysis, and colorimetric or fluorescent protein meltinganalyses. Dissociation constants (K_(D)) of the anti-C1q antibodies forC1q may be determined, e.g., using full-length antibodies or antibodyfragments, such as Fab fragments.

The anti-C1q antibodies of this disclosure may bind to C1q antigensderived from any organism having a complement system, including anymammalian organism such as human, mouse, rat, rabbit, monkey, dog, cat,cow, horse, camel, sheep, goat, or pig. In some embodiments, theanti-C1q antibodies bind specifically to epitopes on human C1q. In someembodiments, the anti-C1q antibodies specifically bind to epitopes onboth human and mouse C1q. In some embodiments, the anti-C1q antibodiesspecifically bind to epitopes on human, mouse, and rat C1q.

In some embodiments, the anti-C1q antibody inhibits the interactionbetween C1q and an autoantibody. In certain embodiments, theautoantibody recognizes gangliosides (e.g., anti-GQ1b). In someembodiments, the anti-C1q antibody inhibits the interaction between C1qand C1r, or between C1q and C1s, or between C1q and both C1r and C1s. Insome embodiments, the anti-C1q antibody binds to the C1q A-chain. Inother embodiments, the anti-C1q antibody binds to the C1q B-chain. Inother embodiments, the anti-C1q antibody binds to the C1q C-chain. Insome embodiments, the anti-C1q antibody binds to the globular domain ofthe C1q A-, B-, or C-chain. In other embodiments, the anti-C1q antibodybinds to the collagen-like domain of the C1q A-, B-, or C-chain.

In some embodiments, provided herein is an anti-C1q antibody that bindsto an epitope of C1q that is the same as or overlaps with the C1qepitope bound by another antibody of this disclosure. In certainembodiments, provided herein is an anti-C1q antibody that binds to anepitope of C1q that is the same as or overlaps with the C1q epitopebound by anti-C1q antibody M1. In some embodiments, the anti-C1qantibody competes with another antibody of this disclosure for bindingto C1q. In certain embodiments, the anti-C1q antibody competes withanti-C1q antibody M1 or an antigen-binding fragment thereof for bindingto C1q. Methods that may be used to determine which C1q epitope of ananti-C1q antibody binds to, or whether two antibodies bind to the sameor an overlapping epitope, may include, without limitation, X-raycrystallography, NMR spectroscopy, Alanine-Scanning Mutagenesis, thescreening of peptide libraries that include C1q-derived peptides withoverlapping C1q sequences, and competition assays.

In some embodiments, provided herein are anti-C1q antibodies thatcompete with antibody M1 or an anti-C1q antibody described herein forbinding to C1q. Competition assays can be used to determine whether twoantibodies bind the same epitope by recognizing identical or stericallyoverlapping epitopes or one antibody competitively inhibits binding ofanother antibody to the antigen. These assays are known in the art.Typically, antigen or antigen expressing cells is immobilized on amulti-well plate and the ability of unlabeled antibodies to block thebinding of labeled antibodies is measured. Common labels for suchcompetition assays are radioactive labels or enzyme labels.

Competitive anti-C1q antibodies encompassed herein are antibodies thatinhibit (i.e., prevent or interfere with in comparison to a control) orreduce the binding of any anti-C1q antibody of this disclosure (such asM1 or an antigen-binding fragment of M1) to C1q by at least 50%, 60%,70%, 80%, 90% and 95% at 1 μM or less. For example, the concentrationcompeting antibody in the competition assay may be at or below the K_(D)of antibody M1 or an antigen-binding fragment of M1. Competition betweenbinding members may be readily assayed in vitro for example using ELISAand/or by monitoring the interaction of the antibodies with C1q insolution. The exact means for conducting the analysis is not critical.C1q may be immobilized to a 96-well plate or may be placed in ahomogenous solution. In specific embodiments, the ability of unlabeledcandidate antibody(ies) to block the binding of the labeled anti-C1qantibody, e.g. M1, can be measured using radioactive, enzyme or otherlabels. In the reverse assay, the ability of unlabeled antibodies tointerfere with the interaction of a labeled anti-C1q antibody with C1qwherein said labeled anti-C1q antibody, e.g., M1, and C1q are alreadybound is determined. The readout is through measurement of bound label.C1q and the candidate antibody(ies) may be added in any order or at thesame time.

In some embodiments, the anti-C1q antibody inhibits the interactionbetween C1q and an autoantibody. In some embodiments, the anti-C1qantibody is murine anti-human C1q monoclonal antibody M1, which isproduced by a hybridoma cell line deposited with ATCC on Jun. 6, 2013with ATCC Accession Number PTA-120399.

In some embodiments, the anti-C1q antibody is an isolated antibody whichbinds essentially the same C1q epitope as M1. In some embodiments, theanti-C1q antibody is an isolated antibody comprising the HVR-L1, HVR-L2,and HVR-L3 of the light chain variable domains of monoclonal antibody M1produced by the hybridoma cell line deposited with ATCC on Jun. 6, 2013with ATCC Accession Number PTA-120399, or progeny thereof. In someembodiments, the anti-C1q antibody is an isolated antibody comprisingthe HVR-H1, HVR-H2, and HVR-H3 of the heavy chain variable domains ofmonoclonal antibody M1 produced by the hybridoma cell line depositedwith ATCC on Jun. 6, 2013 with ATCC Accession Number PTA-120399, orprogeny thereof. In some embodiments, the anti-C1q antibody is anisolated antibody comprising the HVR-L1, HVR-L2, and HVR-L3 of the lightchain variable domains and the HVR-H1, HVR-H2, and HVR-H3 of the heavychain variable domains of monoclonal antibody M1 produced by thehybridoma cell line deposited with ATCC on Jun. 6, 2013 with ATCCAccession Number PTA-120399, or progeny thereof.

In some embodiments, the anti-C1q antibody binds to a C1q protein andbinds to one or more amino acids of the C1q protein within amino acidresidues selected from (a) amino acid residues 196-226 of SEQ ID NO:1,or amino acid residues of a C1q protein chain A (C1 qA) corresponding toamino acid residues 196-226 (GLFQVVSGGMVLQLQQGDQVWVEKDPKKGHI) of SEQ IDNO:1; (b) amino acid residues 196-221 of SEQ ID NO:1, or amino acidresidues of a C1qA corresponding to amino acid residues 196-221(GLFQVVSGGMVLQLQQGDQVWVEKDP) of SEQ ID. NO:1; (c) amino acid residues202-221 of SEQ ID NO:1, or amino acid residues of a C1qA correspondingto amino acid residues 202-221 (SGGMVLQLQQGDQVWVEKD) of SEQ ID NO:1; (d)amino acid residues 202-219 of SEQ ID NO:1, or amino acid residues of aC1qA corresponding to amino acid residues 202-219 (SGGMVLQLQQGDQVWVEK)of SEQ ID NO:1; and (e) amino acid residues Lys 219 and/or Ser 202 ofSEQ ID NO:1, or amino acid residues of a C1qA corresponding Lys 219and/or Ser 202 of SEQ ID NO:1.

In some embodiments, the antibody further binds to one or more aminoacids of the C1q protein within amino acid residues selected from: (a)amino acid residues 218-240 of SEQ ID NO:3 or amino acid residues of aC1q protein chain C (C1qC) corresponding to amino acid residues 218-240(WLAVNDYYDMVGI QGSDSVFSGF) of SEQ ID NO:3; (b) amino acid residues225-240 of SEQ ID NO:3 or amino acid residues of a C1qC corresponding toamino acid residues 225-240 (YDMVGI QGSDSVFSGF) of SEQ ID NO:3; (c)amino acid residues 225-232 of SEQ ID NO:3 or amino acid residues of aC1qC corresponding to amino acid residues 225-232 (YDMVGIQG) of SEQ IDNO:3; (d) amino acid residue Tyr 225 of SEQ ID NO:3 or an amino acidresidue of a C1qC corresponding to amino acid residue Tyr 225 of SEQ IDNO:3; (e) amino acid residues 174-196 of SEQ ID NO:3 or amino acidresidues of a C1qC corresponding to amino acid residues 174-196(HTANLCVLLYRSGVKVVTFCGHT) of SEQ ID NO:3; (f) amino acid residues184-192 of SEQ ID NO:3 or amino acid residues of a C1qC corresponding toamino acid residues 184-192 (RSGVKVVTF) of SEQ ID NO:3; (g) amino acidresidues 185-187 of SEQ ID NO:3 or amino acid residues of a C1qCcorresponding to amino acid residues 185-187 (SGV) of SEQ ID NO:3; (h)amino acid residue Ser 185 of SEQ ID NO:3 or an amino acid residue of aC1qC corresponding to amino acid residue Ser 185 of SEQ ID NO:3.

In certain embodiments, the anti-C1q antibody binds to amino acidresidue Lys 219 and Ser 202 of the human C1qA as shown in SEQ ID NO:1 oramino acids of a human C1qA corresponding to Lys 219 and Ser 202 asshown in SEQ ID NO:1, and amino acid residue Tyr 225 of the human C1qCas shown in SEQ ID NO:3 or an amino acid residue of a human C1qCcorresponding to Tyr 225 as shown in SEQ ID NO:3. In certainembodiments, the anti-C1q antibody binds to amino acid residue Lys 219of the human C1qA as shown in SEQ ID NO:1 or an amino acid residue of ahuman C1qA corresponding to Lys 219 as shown in SEQ ID NO:1, and aminoacid residue Ser 185 of the human C1qC as shown in SEQ ID NO:3 or anamino acid residue of a human C1qC corresponding to Ser 185 as shown inSEQ ID NO:3.

In some embodiments, the anti-C1q antibody binds to a C1q protein andbinds to one or more amino acids of the C1q protein within amino acidresidues selected from: (a) amino acid residues 218-240 of SEQ ID NO:3or amino acid residues of a C1qC corresponding to amino acid residues218-240 (WLAVNDYYDMVGI QGSDSVFSGF) of SEQ ID NO:3; (b) amino acidresidues 225-240 of SEQ ID NO:3 or amino acid residues of a C1qCcorresponding to amino acid residues 225-240 (YDMVGI QGSDSVFSGF) of SEQID NO:3; (c) amino acid residues 225-232 of SEQ ID NO:3 or amino acidresidues of a C1qC corresponding to amino acid residues 225-232(YDMVGIQG) of SEQ ID NO:3; (d) amino acid residue Tyr 225 of SEQ ID NO:3or an amino acid residue of a C1qC corresponding to amino acid residueTyr 225 of SEQ ID NO:3; (e) amino acid residues 174-196 of SEQ ID NO:3or amino acid residues of a C1qC corresponding to amino acid residues174-196 (HTANLCVLLYRSGVKVVTFCGHT) of SEQ ID NO:3; (f) amino acidresidues 184-192 of SEQ ID NO:3 or amino acid residues of a C1qCcorresponding to amino acid residues 184-192 (RSGVKVVTF) of SEQ ID NO:3;(g) amino acid residues 185-187 of SEQ ID NO:3 or amino acid residues ofa C1qC corresponding to amino acid residues 185-187 (SGV) of SEQ IDNO:3; (h) amino acid residue Ser 185 of SEQ ID NO:3 or an amino acidresidue of a C1qC corresponding to amino acid residue Ser 185 of SEQ IDNO:3.

In some embodiments, the anti-C1q antibody inhibits the interactionbetween C1q and an autoantibody at a stoichiometry of less than 2.5:1;2.0:1; 1.5:1; or 1.0:1. In other embodiments, the anti-C1q antibodybinds to C1q with a stoichiometry of less than 20:1; less than 19.5:1;less than 19:1; less than 18.5:1; less than 18:1; less than 17.5:1; lessthan 17:1; less than 16.5:1; less than 16:1; less than 15.5:1; less than15:1; less than 14.5:1; less than 14:1; less than 13.5:1; less than13:1; less than 12.5:1; less than 12:1; less than 11.5:1; less than11:1; less than 10.5:1; less than 10:1; less than 9.5:1; less than 9:1;less than 8.5:1; less than 8:1; less than 7.5:1; less than 7:1; lessthan 6.5:1; less than 6:1; less than 5.5:1; less than 5:1; less than4.5:1; less than 4:1; less than 3.5:1; less than 3:1; less than 2.5:1;less than 2.0:1; less than 1.5:1; or less than 1.0:1. In certainembodiments, the anti-C1q antibody binds C1q with a bindingstoichiometry that ranges from 20:1 to 1.0:1 or less than 1.0:1. Incertain embodiments, the anti-C1q antibody binds C1q with a bindingstoichiometry that ranges from 6:1 to 1.0:1 or less than 1.0:1. Incertain embodiments, the anti-C1q antibody binds C1q with a bindingstoichiometry that ranges from 2.5:1 to 1.0:1 or less than 1.0:1. Insome embodiments, the anti-C1q antibody inhibits the interaction betweenC1q and C1r, or between C1q and C1s, or between C1q and both C1r andC1s. In some embodiments, the anti-C1q antibody inhibits the interactionbetween C1q and C1r, between C1q and C1s, and/or between C1q and bothC1r and C1s. In some embodiments, the anti-C1q antibody binds to the C1qA-chain. In other embodiments, the anti-C1q antibody binds to the C1qB-chain. In other embodiments, the anti-C1q antibody binds to the C1qC-chain. In some embodiments, the anti-C1q antibody binds to the C1qA-chain, the C1q B-chain and/or the C1q C-chain. In some embodiments,the anti-C1q antibody binds to the globular domain of the C1q A-chain,B-chain, and/or C-chain. In other embodiments, the anti-C1q antibodybinds to the collagen-like domain of the C1q A-chain, the C1q B-chain,and/or the C1q C-chain.

In some embodiments, the anti-C1q antibodies of this disclosure inhibitC1F hemolysis (also referred to as CH50 hemolysis) by at least 20%, atleast 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 95%, or at least 99%, or by an amountthat ranges from at least 30% to at least 99%, relative to a controlwherein the anti-C1q antibodies of this disclosure are absent or whereincontrol antibodies are used that do not bind to a complement factor oranother antibody such as an autoantibody. Methods for measuring C1Fhemolysis are well known in the art. The EC₅₀ values for anti-C1qantibodies of this disclosure with respect to C1F hemolysis may be lessthan 3 μg/ml; 2.5 μg/ml; 2.0 μg/ml; 1.5 μg/ml; 1.0 μg/ml; 0.5 μg/ml;0.25 μg/ml; 0.1 μg/ml; 0.05 μg/ml. In some embodiments, the anti-C1qantibodies of this disclosure neutralize at least 50% of C1F hemolysisat a dose of less than 200 ng/ml, less than 100 ng/ml, less than 50ng/ml, or less than 20 ng/ml. In some embodiments, the anti-C1qantibodies of this disclosure neutralize C1F hemolysis at approximatelyequimolar concentrations of C1q and the anti-C1q antibody. In someembodiments, the anti-C1q antibodies of this disclosure neutralizehemolysis in a human C1F hemolysis assay. In some embodiments, theanti-C1q antibodies of this disclosure neutralize hemolysis in a human,mouse, and rat C1F hemolysis assay.

In some embodiments, the alternative pathway may amplify CDC initiatedby C1q binding and subsequent C1s activation; in at least some of theseembodiments, the anti-C1q antibodies of this disclosure inhibit thealternative pathway by at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, at least 95%, or atleast 99%, or by an amount that ranges from at least 30% to at least99%, relative to a control wherein the anti-C1q antibodies of thisdisclosure are absent.

Anti-C1s Antibodies

The anti-C1s antibodies of this disclosure recognize and bind tocomplement factor C1s and/or C1s in the C1 complex of the classicalcomplement activation pathway.

In some embodiments, the anti-C1s antibodies neutralize an activity ofcomplement factor C1s. In some embodiments, the anti-C1s antibodiesinhibit the interaction between complement factor C1s and othercomplement factors, such as C1q or C1r, or complement proteasesubstrates, such as C4. In some embodiments, the anti-C1s antibodiesinhibit the catalytic activity of the serine protease C1s or inhibit theprocessing of a serine protease pro-form to an active protease. In someembodiments the anti-C1s antibodies inhibit the classical pathway. Incertain embodiments the antibodies further inhibit the alternativepathway. In some embodiments, the anti-C1s antibodies inhibitautoantibody- and complement-dependent cytotoxicity (CDC). In someembodiments, the anti-C1s antibodies inhibit complement-dependentcell-mediated cytotoxicity (CDCC).

The functional properties of the anti-C1s antibodies of this invention,such as dissociation constants for antigens, inhibition ofprotein-protein interactions (e.g., C1s-C1q interactions), inhibition ofautoantibody-dependent and complement-dependent cytotoxicity (CDC),inhibition of complement-dependent cell-mediated cytotoxicity (CDCC), orlesion formation, may, without limitation, be measured in in vitro, exvivo, or in vivo experiments.

The dissociation constants (K_(D)) of the anti-C1s antibodies for C1smay be less than 100 nM, less than 90 nM, less than 80 nM, less than 70nM, less than 60 nM, less than 50 nM, less than 40 nM, less than 30 nM,less than 20 nM, less than 10 nM, less than 9 nM, less than 8 nM, lessthan 7 nM, less than 6 nM, less than 5 nM, less than 4 nM, less than 3nM, less than 2 nM, less than 1 nM, less than 0.5 nM, less than 0.1 nM,less than 0.05 nM, less than 0.01 nM, or less than 0.005 nM. Preferably,dissociation constants are less than 20 nM. Antibody dissociationconstants for antigens other than C1s may be at least 5-fold, at least10-fold, at least 100-fold, at least 1,000-fold, at least 10,000-fold,or at least 100,000-fold higher that the dissociation constants fortheir respective antigens. For example, the dissociation constant of aC1s antibody of this disclosure may be at least 1,000-fold higher forC1q than for C1s. Dissociation constants may be determined through anyanalytical technique, including any biochemical or biophysical techniquesuch as surface plasmon resonance (SPR), isothermal titrationcalorimetry (ITC), differential scanning calorimetry (DSC), circulardichroism (CD), stopped-flow analysis, and colorimetric or fluorescentprotein melting analyses. Dissociation constants (K_(D)) of the anti-C1santibodies for their respective antigens may be determined, e.g., usingfull-length antibodies or antibody fragments, such as Fab fragments. Theantibodies of this disclosure may bind to C1s antigens derived from anyorganism having a complement system, including any mammalian organismsuch as human, mouse, rat, rabbit, monkey, dog, cat, cow, horse, camel,sheep, goat, or pig. In preferred embodiments, the antibodies of thisdisclosure bind to epitopes comprising amino acid residues on human C1s.

In some embodiments, provided herein are anti-C1s antibodies thatcompete with antibody 5A1 or 5C12, or an anti-C1s antibody describedherein for binding to C1s. Competition assays can be used to determinewhether two antibodies bind the same epitope by recognizing identical orsterically overlapping epitopes or one antibody competitively inhibitsbinding of another antibody to the antigen. These assays are known inthe art. Typically, antigen or antigen expressing cells is immobilizedon a multi-well plate and the ability of unlabeled antibodies to blockthe binding of labeled antibodies is measured. Common labels for suchcompetition assays are radioactive labels or enzyme labels.

Competitive anti-C1s antibodies encompassed herein are antibodies thatinhibit (i.e., prevent or interfere with in comparison to a control) orreduce 5A1, 5C12, or an anti-C1s antibody described herein binding toC1s by at least 50%, 60%, 70%, and 80% in order of increasing preference(even more preferably, at least 90% and, most preferably, at least 95%)at 1 μM or less with 5A1, 5C12, or an anti-C1s antibody described hereinat or below its K_(D). Competition between binding members may bereadily assayed in vitro for example using ELISA and/or by monitoringthe interaction of the antibodies with C1s in solution. The exact meansfor conducting the analysis is not critical. C1s may be immobilized to a96-well plate or may be placed in a homogenous solution. In specificembodiments, the ability of unlabeled candidate antibody or antibodiesto block the binding of labeled 5A1 or 5C12 can be measured usingradioactive, enzyme, or other labels. In the reverse assay, the abilityof unlabeled antibodies to interfere with the interaction of labeled 5A1or 5C12 with C1s wherein said 5A1 or 5C12 and C1s are already bound isdetermined. The readout is through measurement of bound label. C1s andthe candidate antibody or antibodies may be added in any order or at thesame time.

In some preferred embodiments, the anti-C1s antibody is murineanti-human C1s monoclonal antibody 5A1, which is produced by a hybridomacell line deposited with ATCC on May 15, 2013 having ATCC AccessionNumber PTA-120351. In some embodiments, the anti-C1s antibody is anisolated antibody which binds essentially the same C1s epitope as 5A1.In some embodiments, the anti-C1s antibody is an isolated antibodycomprising the HVR-L1, HVR-L2, and HVR-L3 of the light chain variabledomains of monoclonal antibody 5A1 produced by the hybridoma cell linedeposited with ATCC on May 15, 2013 having ATCC Accession NumberPTA-120351, or progeny thereof. In some embodiments, the anti-C1santibody is an isolated antibody comprising the HVR-H1, HVR-H2, andHVR-H3 of the heavy chain variable domains of monoclonal antibody 5A1produced by the hybridoma cell line deposited with ATCC on May 15, 2013having ATCC Accession Number PTA-120351, or progeny thereof. In someembodiments, the anti-C1s antibody is an isolated antibody comprisingthe HVR-L1, HVR-L2, and HVR-L3 of the light chain variable domains andthe HVR-H1, HVR-H2, and HVR-H3 of the heavy chain variable domains ofmonoclonal antibody 5A1 produced by the hybridoma cell line depositedwith ATCC on May 15, 2013 having ATCC Accession Number PTA-120351, orprogeny thereof.

In some preferred embodiments, the anti-C1s antibody is murineanti-human C1s monoclonal antibody 5C12, which is produced by ahybridoma cell line deposited with ATCC on May 15, 2013 having ATCCAccession Number PTA-120352. In some embodiments, the anti-C1s antibodyis an isolated antibody which binds essentially the same C1s epitope as5C12. In some embodiments, the anti-C1s antibody is an isolated antibodycomprising the HVR-L1, HVR-L2, and HVR-L3 of the light chain variabledomains of monoclonal antibody 5C12 produced by the hybridoma cell linedeposited with ATCC on May 15, 2013 having ATCC Accession NumberPTA-120352, or progeny thereof. In some embodiments, the anti-C1santibody is an isolated antibody comprising the HVR-H1, HVR-H2, andHVR-H3 of the heavy chain variable domains of monoclonal antibody 5C12produced by the hybridoma cell line deposited with ATCC on May 15, 2013having ATCC Accession Number PTA-120352, or progeny thereof. In someembodiments, the anti-C1s antibody is an isolated antibody comprisingthe HVR-L1, HVR-L2, and HVR-L3 of the light chain variable domains andthe HVR-H1, HVR-H2, and HVR-H3 of the heavy chain variable domains ofmonoclonal antibody 5A1 produced by the hybridoma cell line depositedwith ATCC on May 15, 2013 having ATCC Accession Number PTA-120352, orprogeny thereof.

In some embodiments, the anti-C1s antibody inhibits the interactionbetween C1s and C1q. In some embodiments, the anti-C1s antibody inhibitsthe interaction between C1s and C1r. In some embodiments the anti-C1santibody inhibits the interaction between C1s and C1q and between C1sand C1r. In some embodiments, the anti-C1s antibody inhibits thecatalytic activity of C1s or the processing of pro-C1s to an activeprotease. In some embodiments, the anti-C1s antibody inhibits theinteraction between C1s and its substrates such as C2 and C4. In someembodiments, the anti-C1s antibody binds to C1s respective interactions,at a stoichiometry of less than 2.5:1; 2.0:1; 1.5:1; or 1.0:1. In someembodiments, the anti-C1s antibody binds to C1s with a stoichiometry ofless than 20:1; less than 19.5:1; less than 19:1; less than 18.5:1; lessthan 18:1; less than 17.5:1; less than 17:1; less than 16.5:1; less than16:1; less than 15.5:1; less than 15:1; less than 14.5:1; less than14:1; less than 13.5:1; less than 13:1; less than 12.5:1; less than12:1; less than 11.5:1; less than 11:1; less than 10.5:1; less than10:1; less than 9.5:1; less than 9:1; less than 8.5:1; less than 8:1;less than 7.5:1; less than 7:1; less than 6.5:1; less than 6:1; lessthan 5.5:1; less than 5:1; less than 4.5:1; less than 4:1; less than3.5:1; less than 3:1; less than 2.5:1; less than 2.0:1; less than 1.5:1;or less than 1.0:1. In certain embodiments, the anti-C1s antibody bindsC1s with a binding stoichiometry that ranges from 20:1 to 1.0:1 or lessthan 1.0:1. In certain embodiments, the anti-C1s antibody binds C1s witha binding stoichiometry that ranges from 6:1 to 1.0:1 or less than1.0:1. In certain embodiments, the anti-C1s antibody binds C1s with abinding stoichiometry that ranges from 2.5:1 to 1.0:1 or less than1.0:1. In preferred embodiments, the C1s antibody inhibits aninteraction, such as the C1s-C4 interaction, at approximately equimolarconcentrations of C1s and the anti-C1s antibody. In some embodiments theanti-C1s antibody inhibits activation of C1s, C1r, or of both C1s andC1r.

In some embodiments, the anti-C1s antibodies of this disclosure inhibitC1F hemolysis by at least 20%, at least 30%, at least 40%, at least 50%,at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, orat least 99% relative to a control wherein the anti-C1s antibodies ofthis disclosure are absent (see also Example 3). In certain embodiments,anti-C1s antibodies of this disclosure inhibit C1F hemolysis by anamount that ranges from at least 30% to at least 99% relative to acontrol wherein the antibodies of this disclosure are absent. Methodsfor measuring C1F hemolysis are well known in the art (see also Example3 for possible methods). The EC₅₀ values for antibodies of thisdisclosure with respect to C1F hemolysis may be less than less than 3μg/ml; less than 2.5 μg/ml; less than 2.0 μg/ml; less than 1.5 μg/ml;less than 1.0 μg/ml; less than 0.5 μg/ml; less than 0.25 μg/ml; lessthan 0.1 μg/ml; or less than 0.05 μg/ml. Preferably, EC₅₀ values areless than 1.0 μg/ml (see also Example 3). Preferably, the anti-C1santibodies of this disclosure inhibit C1F hemolysis at approximatelyequimolar concentrations of C1s and the respective anti-C1s antibody.

In some embodiments, the alternative pathway may amplify CDC initiatedby C1q binding and subsequent C1s activation; in at least some of theseembodiments, the anti-C1s antibodies of this disclosure inhibit thealternative pathway by at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, at least 95%, or atleast 99% relative to a control wherein the antibodies of thisdisclosure are absent. In certain embodiments, anti-C1s antibodies ofthis disclosure inhibit the alternative pathway by an amount that rangesfrom at least 30% to at least 99% relative to a control wherein theanti-C1s antibodies of this disclosure are absent.

In some embodiments, the anti-C1s antibodies of this disclosure preventlesion formation in an ex vivo spinal cord slice model of NMO or in anin vivo mouse model of NMO. Methods for measuring lesion formation exvivo or in vivo are well known in the art. Ex vivo lesion formation maybe reduced at least by a relative score of 0.5, 1.0, 1.5, 2.0, 2.5, 3.0,3.5, or 4.0. Preferably, ex vivo lesion formation is reduced by arelative score of at least 2.5. The EC₅₀ values for anti-C1s antibodiesof this disclosure with respect to the prevention of ex vivo lesionformation may be less than 3 μg/ml; less than 2.5 μg/ml; less than 2.0μg/ml; less than 1.5 μg/ml; less than 1.0 μg/ml; less than 0.5 μg/ml;less than 0.25 μg/ml; less than 0.1 μg/ml; or less than 0.05 μg/ml.Preferably, EC₅₀ values are less than 1.0 μM. In vivo lesion formationmay be reduced by at least 5%, at least 10%, at least 15%, at least 20%,at least 35%, at least 40%, or at least 50% in terms of loss of staining(% of area). Staining may be assessed, without limitation, by AQP4staining, GFAP staining, or MBP staining Preferably, in vivo lesionformation is reduced by at least 10%.

Anti-C1r Antibodies

The anti-C1r antibodies of this disclosure recognize and bind tocomplement factor C1r and/or C1r in the C1 complex of the classicalcomplement activation pathway.

In some embodiments, the anti-C1r antibodies neutralize an activity ofcomplement factor C1r. In some embodiments that anti-C1r antibodyinhibits the interaction between C1r and C1q. In some embodiments, theanti-C1r antibody inhibits the interaction between C1r and C1s. In someembodiments, the anti-C1r antibody inhibits the interaction between C1rand C1q and between C1r and C1s. In some embodiments, the anti-C1rantibody inhibits the catalytic activity of C1r and/or the processing ofpro-C1r to an active protease. In some embodiments, the anti-C1rantibody inhibits the respective interactions at a stoichiometry of lessthan 2.5:1; 2.0:1; 1.5:1; or 1.0:1. In some embodiments, the anti-C1rantibody binds to C1r with a stoichiometry of less than 20:1; less than19.5:1; less than 19:1; less than 18.5:1; less than 18:1; less than17.5:1; less than 17:1; less than 16.5:1; less than 16:1; less than15.5:1; less than 15:1; less than 14.5:1; less than 14:1; less than13.5:1; less than 13:1; less than 12.5:1; less than 12:1; less than11.5:1; less than 11:1; less than 10.5:1; less than 10:1; less than9.5:1; less than 9:1; less than 8.5:1; less than 8:1; less than 7.5:1;less than 7:1; less than 6.5:1; less than 6:1; less than 5.5:1; lessthan 5:1; less than 4.5:1; less than 4:1; less than 3.5:1; less than3:1; less than 2.5:1; less than 2.0:1; less than 1.5:1; or less than1.0:1. In certain embodiments, the anti-C1r antibody binds C1r with abinding stoichiometry that ranges from 20:1 to 1.0:1 or less than 1.0:1.In certain embodiments, the anti-C1r antibody binds C1r with a bindingstoichiometry that ranges from 6:1 to 1.0:1 or less than 1.0:1. Incertain embodiments, the anti-C1r antibody binds C1r with a bindingstoichiometry that ranges from 2.5:1 to 1.0:1 or less than 1.0:1. Insome embodiments the anti-C1r antibody inhibits activation of C1r, C1s,and/or both C1r and C1s. Activation of the serine proteases C1r and C1scan be measured, without limitation, by standard colorimetric orfluorescent serine protease assays or by a standard complement-mediatedcell lysis assays that are well known in the art.

Anti-C1 Complex Antibodies

The anti-C1 complex antibodies of this disclosure recognize and bind toC1 complex and/or complement factors C1q, C1s, and/or C1r in the C1complex of the classical complement activation pathway.

In some embodiments, the anti-C1 complex antibodies neutralize anactivity of C1 complex, complement factor C1q, complement factor C1s,and/or complement factor C1r. In some embodiments the anti-C1 complexantibody inhibits the interaction between C1q and C1s, C1q and C1r,and/or C1s and C1r. In some embodiments, the anti-C1 complex antibodyinhibits C1r or C1s activation or prevents their ability to act on C2 orC4. In some embodiments, the anti-C1 complex antibody inhibits therespective interactions at a stoichiometry of less than 2.5:1; 2.0:1;1.5:1; or 1.0:1. In some embodiments, the anti-C1 complex antibody bindsto the C1 complex with a stoichiometry of less than 20:1; less than19.5:1; less than 19:1; less than 18.5:1; less than 18:1; less than17.5:1; less than 17:1; less than 16.5:1; less than 16:1; less than15.5:1; less than 15:1; less than 14.5:1; less than 14:1; less than13.5:1; less than 13:1; less than 12.5:1; less than 12:1; less than11.5:1; less than 11:1; less than 10.5:1; less than 10:1; less than9.5:1; less than 9:1; less than 8.5:1; less than 8:1; less than 7.5:1;less than 7:1; less than 6.5:1; less than 6:1; less than 5.5:1; lessthan 5:1; less than 4.5:1; less than 4:1; less than 3.5:1; less than3:1; less than 2.5:1; less than 2.0:1; less than 1.5:1; or less than1.0:1. In certain embodiments, the anti-C1 complex antibody binds the C1complex with a binding stoichiometry that ranges from 20:1 to 1.0:1 orless than 1.0:1. In certain embodiments, the anti-C1 complex antibodybinds the C1 complex with a binding stoichiometry that ranges from 6:1to 1.0:1 or less than 1.0:1. In certain embodiments, the anti-C1 complexantibody binds the C1 complex with a binding stoichiometry that rangesfrom 2.5:1 to 1.0:1 or less than 1.0:1.

Antibody Characteristics

Where antibodies of this disclosure inhibit the interaction between twoor more complement factors, such as the interaction of C1q and C1s, orthe interaction between factor C1q and an autoantibody, the interactionoccurring in the presence of the antibody is reduced by at least 10%, atleast 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, at least 99%, or atleast 99.9% compared to the interaction occurring in the absence of theantibody, but otherwise identical conditions. In certain embodiments,the interaction occurring in the presence of the antibody is reduced byan amount that ranges from at least 30% to at least 99.9%. Whereantibodies of this disclosure inhibit activation of C1s and/or C1r, theserine protease activity of C1s and/or C1r in the presence of theantibody is reduced by at least 10%, at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, at least 99%, or at least 99.9% compared to theserine protease activity in the absence of the antibody. In certainembodiments, the serine protease activity of C1s and/or C1r in thepresence of the antibody in the presence of the antibody is reduced byan amount that ranges from at least 30% to at least 99.9%.

In some embodiments, the antibodies of this disclosure inhibitC4-cleavage by at least 20%, at least 30%, at least 40%, at least 50%,at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, orat least 99% relative to a control wherein the antibodies of thisdisclosure are absent. In certain embodiments, the antibodies of thisdisclosure inhibit C4-cleavage by an amount that ranges from at least30% to at least 99% relative to a control wherein the antibodies of thisdisclosure are absent. Methods for measuring C4-cleavage are well knownin the art (see also Example 3 for possible methods). The EC₅₀ valuesfor antibodies of this disclosure with respect C4-cleavage may be lessthan 3 μg/ml; less than 2.5 μg/ml; less than 2.0 μg/ml; less than 1.5μg/ml; less than 1.0 μg/ml; less than 0.5 μg/ml; less than 0.25 μg/ml;less than 0.1 μg/ml; or less than 0.05 μg/ml. Preferably, EC₅₀ valuesare less than 1.0 μg/ml. Preferably, the antibodies of this disclosureinhibit C4-cleavage at approximately equimolar concentrations of C1s andthe respective anti-C1s antibody.

In some embodiments, the antibodies of this disclosure inhibitautoantibody-dependent and complement-dependent cytotoxicity (CDC) by atleast 10%, at least 20%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, at least 95%, atleast 99%, or at least 99.9% relative to a control wherein theantibodies of the present disclosure are absent. In certain embodiments,antibodies of the present disclosure inhibit CDC by an amount thatranges from at least 30% to at least 99.9%. The EC₅₀ values forantibodies of this disclosure, e.g., with respect to inhibition ofautoantibody-dependent and complement-dependent cytotoxicity (CDC), maybe less than 3 μg/ml; less than 2.5 μg/ml; 2.0 μg/ml; less than 1.5μg/ml; less than 1.0 μg/ml; less than 0.5 μg/ml; less than 0.25 μg/ml;less than 0.1 μg/ml; or less than 0.05 μg/ml. Preferably, EC₅₀ valuesare less than 1.0 μg/ml (see also Example 3). In certain embodiments,the EC₅₀ values for antibodies of this disclosure range from 3 μg/ml to0.05 μg/ml or less than 0.05 μg/ml. In some embodiments, the EC₅₀ valuesfor antibodies of the present disclosure may be calculated in thepresence of human complement, for example present in human serum. Insome embodiments, the amount of human complement, for example humanserum, ranges from less than 1% to at least 20%. In certain embodiments,the amount of human complement, for example human serum, is less than1%. In certain embodiments, the amount of human complement, for examplehuman serum, is at least 1%, at least 2%, at least 3%, at least 4% atleast 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least10%, at least 11%, at least 12%, at least 13%, at least 14%, at least15%, at least 16%, at least 17%, at least 18%, at least 19%, or at least20%.

In some embodiments, the antibodies of this disclosure inhibitcomplement-dependent cell-mediated cytotoxicity (CDCC) by at least 10%,at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, at least 99%, or atleast 99.9% relative to a control wherein the antibodies of the presentdisclosure are absent. In certain embodiments, antibodies of the presentdisclosure inhibit CDCC by an amount that ranges from at least 30% to atleast 99.9%. Methods for measuring CDCC are well known in the art (seealso Example 5 for possible methods). The EC₅₀ values for antibodies ofthis disclosure, for example, with respect CDCC inhibition may be lessthan 3 μg/ml; less than 2.5 μg/ml; 2.0 μg/ml; less than 1.5 μg/ml; lessthan 1.0 μg/ml; less than 0.5 μg/ml; less than 0.25 μg/ml; less than 0.1μg/ml; or less than 0.05 μg/ml. Preferably, EC₅₀ values are less than1.0 μg/ml. In certain embodiments, the EC₅₀ values for antibodies of thepresent disclosure range from 3 μg/ml to 0.05 μg/ml or less than 0.05μg/ml. In some embodiments, the EC₅₀ values for antibodies of thepresent disclosure may be calculated in the presence of humancomplement, for example present in human serum. In some embodiments, theamount of human complement, for example human serum, ranges from lessthan 1% to at least 20%. In certain embodiments, the amount of humancomplement, for example human serum, is less than 1%. In certainembodiments, the amount of human complement, for example human serum, isat least 1%, at least 2%, at least 3%, at least 4% at least 5%, at least6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%,at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, atleast 17%, at least 18%, at least 19%, or at least 20%. In preferredembodiments, the antibodies of the present disclosure inhibit CDCC butnot antibody-dependent cellular cytotoxicity (ADCC; see also Example 5).In some embodiments, the antibodies of the present disclosure do notinhibit the lectin complement activation pathway.

As disclosed herein, the alternative pathway may amplify CDC initiatedby C1 complex, e.g., by C1q binding to an autoantibody such as anti-GQ1bautoantibodies. Accordingly, in some of embodiments, the antibodies ofthe present disclosure may inhibit the alternative pathway by at least10%, at least 20%, at least 30%, at least 40%, at least 50%, at least60%, at least 70%, at least 80%, at least 90%, at least 95%, at least99%, or at least 99.9% relative to a control wherein the antibodies ofthe present disclosure are absent (see also Example 5). In certainembodiments, antibodies of the present disclosure inhibit thealternative pathway by an amount that ranges from at least 30% to atleast 99.9%.

Additional anti-C1q, anti-C1s, anti-C1r and/or anti-C1 complexantibodies, e.g., antibodies that specifically bind to a C1q, C1s, orC1r protein, or the C1 complex of the present disclosure, may beidentified, screened, and/or characterized for their physical/chemicalproperties and/or biological activities by various assays known in theart.

Additional anti-C1q, anti-C1s, anti-C1r, or anti-C1 complex antibodies,e.g., antibodies that specifically bind to a C1q, C1s, or C1r protein,or the C1 complex of the present disclosure, may be identified,screened, and/or characterized for their physical/chemical propertiesand/or biological activities by various assays known in the art.

In some embodiments, the present disclosure provides anti-C1q, anti-C1s,anti-C1r and/or anti-C1 complex antibodies. The antibodies of thisdisclosure may have one or more of the following characteristics. Theantibodies of this disclosure may be polyclonal antibodies, monoclonalantibodies, human antibodies, humanized antibodies, chimeric antibodies,antibody fragments (e.g., Fab, Fab′-SH, Fv, scFv, and (Fab′)₂fragments), bispecific and polyspecific antibodies, multivalentantibodies, and heteroconjugate antibodies. The antibodies of thisdisclosure may further contain engineered effector functions, amino acidsequence modifications or other antibody modifications known in the art;e.g., the constant region of the anti-C1q, anti-C1s, C1r and/or anti-C1complex antibodies described herein may be modified to impair complementactivation.

In certain embodiment, antibodies of the present disclosure arebispecific antibodies recognizing a first antigen and a second antigen.In some embodiments, the first antigen is a C1q antigen, a C1s antigen,a C1r antigen, and/or a C1 complex antigen. In some embodiments, thesecond antigen is an antigen facilitating transport across theblood-brain-barrier, including without limitation, transferrin receptor(TR), insulin receptor (HIR), insulin-like growth factor receptor(IGFR), low-density lipoprotein receptor related proteins 1 and 2 (LPR-1and 2), diphtheria toxin receptor, CRM197, a llama single domainantibody, TMEM 30(A), a protein transduction domain, TAT, Syn-B,penetratin, a poly-arginine peptide, an angiopep peptide, and ANG1005.

In some embodiments, the antibodies of this disclosure prevent GBS, orone or more symptoms of GBS. In certain embodiments, prevention of GBSor one or more symptoms of GBS by the antibodies of the presentdisclosure is measure by inhibition of C3c deposition, inhibition of MACdepiction, inhibition of axonal damage formation, and/or inhibition ofrespiratory muscle damage in an ex vivo model of GBS, or by an in vivomouse model of GBS. In some embodiments, the antibodies of thisdisclosure inhibit C3c deposition, MAC deposition, axonal damageformation, and/or respiratory muscle damage in an ex vivo model of GBSor in an in vivo mouse model of GBS. Methods for measuring C3cdeposition, MAC deposition, axonal damage formation, and/or respiratorymuscle damage ex vivo or in vivo are well known in the art (see alsoExamples 7-9 for exemplary methods).

Additional anti-C1q, anti-C1s, anti-C1r and anti-C1 complex antibodies,e.g., antibodies that specifically bind to a C1q protein, a C1s protein,a C1r protein, or a C1 complex, of the present disclosure, may beidentified, screened, and/or characterized for their physical/chemicalproperties and/or biological activities by various assays known in theart.

Antibody Preparation

Anti-C1q, anti-C1s, anti-C1r, and/or anti-C1 complex antibodies of thepresent disclosure can encompass polyclonal antibodies, monoclonalantibodies, humanized antibodies, chimeric antibodies, human antibodies,antibody fragments (e.g., Fab, Fab′-SH, Fv, scFv, and F(ab′)₂),bispecific and polyspecific antibodies, multivalent antibodies,heteroconjugate antibodies, library derived antibodies, antibodieshaving modified effector functions, fusion proteins containing anantibody portion, and any other modified configuration of theimmunoglobulin molecule that includes an antigen recognition site, suchas an epitope having amino acid residues of a C1q, C1s, or C1r protein,or the C1 complex of the present disclosure, including glycosylationvariants of antibodies, amino acid sequence variants of antibodies, andcovalently modified antibodies. The anti-C1q, anti-C1s, anti-C1r, and/oranti-C1 complex antibodies may be human, murine, rat, or of any otherorigin (including chimeric or humanized antibodies).

(1) Polyclonal Antibodies

Polyclonal antibodies, such as polyclonal anti-C1q, anti-C1s, anti-C1r,and/or anti-C1 complex antibodies, are generally raised in animals bymultiple subcutaneous (sc) or intraperitoneal (ip) injections of therelevant antigen and an adjuvant. It may be useful to conjugate therelevant antigen (e.g., a purified or recombinant C1q, C1s, or C1rprotein of the present disclosure) to a protein that is immunogenic inthe species to be immunized, e.g., keyhole limpet hemocyanin (KLH),serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor, usinga bifunctional or derivatizing agent, e.g., maleimidobenzoylsulfosuccinimide ester (conjugation through cysteine residues),N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinicanhydride, SOCl₂, or R¹N═C═NR, where R and R¹ are independently loweralkyl groups. Examples of adjuvants which may be employed includeFreund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A,synthetic trehalose dicorynomycolate). The immunization protocol may beselected by one skilled in the art without undue experimentation.

The animals are immunized against the desired antigen, immunogenicconjugates, or derivatives by combining, e.g., 100 μg (for rabbits) or 5μg (for mice) of the protein or conjugate with 3 volumes of Freund'scomplete adjuvant and injecting the solution intradermally at multiplesites. One month later, the animals are boosted with ⅕ to 1/10 theoriginal amount of peptide or conjugate in Freund's complete adjuvant bysubcutaneous injection at multiple sites. Seven to fourteen days later,the animals are bled and the serum is assayed for antibody titer.Animals are boosted until the titer plateaus. Conjugates also can bemade in recombinant-cell culture as protein fusions. Also, aggregatingagents such as alum are suitable to enhance the immune response.

(2) Monoclonal Antibodies

Monoclonal antibodies, such as monoclonal anti-C1q, anti-C1s, anti-C1r,and/or anti-C1 complex antibodies, are obtained from a population ofsubstantially homogeneous antibodies, i.e., the individual antibodiescomprising the population are identical except for possible naturallyoccurring mutations and/or post-translational modifications (e.g.,isomerizations, amidations) that may be present in minor amounts. Thus,the modifier “monoclonal” indicates the character of the antibody as notbeing a mixture of discrete antibodies.

For example, the monoclonal anti-C1q, anti-C1s, anti-C1r, and/or anti-C1complex antibodies may be made using the hybridoma method firstdescribed by Kohler et al., Nature, 256:495 (1975), or may be made byrecombinant DNA methods (U.S. Pat. No. 4,816,567).

In the hybridoma method, a mouse or other appropriate host animal, suchas a hamster, is immunized as hereinabove described to elicitlymphocytes that produce or are capable of producing antibodies thatwill specifically bind to the protein used for immunization (e.g., apurified or recombinant C1q, C1s, or C1r protein of the presentdisclosure). Alternatively, lymphocytes may be immunized in vitro.Lymphocytes then are fused with myeloma cells using a suitable fusingagent, such as polyethylene glycol, to form a hybridoma cell (Goding,Monoclonal Antibodies: Principles and Practice, pp. 59-103 (AcademicPress, 1986)).

The immunizing agent will typically include the antigenic protein (e.g.,a purified or recombinant C1q, C1s, or C1r protein of the presentdisclosure) or a fusion variant thereof. Generally peripheral bloodlymphocytes (“PBLs”) are used if cells of human origin are desired,while spleen or lymph node cells are used if non-human mammalian sourcesare desired. The lymphoctyes are then fused with an immortalized cellline using a suitable fusing agent, such as polyethylene glycol, to forma hybridoma cell. Goding, Monoclonal Antibodies: Principles andPractice, Academic Press (1986), pp. 59-103.

Immortalized cell lines are usually transformed mammalian cells,particularly myeloma cells of rodent, bovine or human origin. Usually,rat or mouse myeloma cell lines are employed. The hybridoma cells thusprepared are seeded and grown in a suitable culture medium thatpreferably contains one or more substances that inhibit the growth orsurvival of the unfused, parental myeloma cells. For example, if theparental myeloma cells lack the enzyme hypoxanthine guaninephosphoribosyl transferase (HGPRT or HPRT), the culture medium for thehybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which are substances that prevent the growth ofHGPRT-deficient-cells.

Preferred immortalized myeloma cells are those that fuse efficiently,support stable high-level production of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. Among these, preferred are murine myeloma lines, such as thosederived from MOPC-21 and MPC-11 mouse tumors (available from the SalkInstitute Cell Distribution Center, San Diego, Calif. USA), as well asSP-2 cells and derivatives thereof (e.g., X63-Ag8-653) (available fromthe American Type Culture Collection, Manassas, Va. USA). Human myelomaand mouse-human heteromyeloma cell lines have also been described forthe production of human monoclonal antibodies (Kozbor, J. Immunol.,133:3001 (1984); Brodeur et al., Monoclonal Antibody ProductionTechniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York,1987)).

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against the antigen (e.g.,a C1q, C1s, or C1r protein of the present disclosure). Preferably, thebinding specificity of monoclonal antibodies produced by hybridoma cellsis determined by immunoprecipitation or by an in vitro binding assay,such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay(ELISA).

The culture medium in which the hybridoma cells are cultured can beassayed for the presence of monoclonal antibodies directed against thedesired antigen (e.g., a C1q, C1s, or C1r protein of the presentdisclosure). Preferably, the binding affinity and specificity of themonoclonal antibody can be determined by immunoprecipitation or by an invitro binding assay, such as radioimmunoassay (RIA) or enzyme-linkedassay (ELISA). Such techniques and assays are known in the in art. Forexample, binding affinity may be determined by the Scatchard analysis ofMunson et al., Anal. Biochem., 107:220 (1980).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, supra). Suitable culture media for this purpose include, forexample, D-MEM or RPMI-1640 medium. In addition, the hybridoma cells maybe grown in vivo as tumors in a mammal.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose chromatography, hydroxylapatitechromatography, gel electrophoresis, dialysis, affinity chromatography,and other methods as described above.

Anti-C1q, anti-C1s, anti-C1r, and/or anti-C1 complex monoclonalantibodies may also be made by recombinant DNA methods, such as thosedisclosed in U.S. Pat. No. 4,816,567, and as described above. DNAencoding the monoclonal antibodies is readily isolated and sequencedusing conventional procedures (e.g., by using oligonucleotide probesthat specifically bind to genes encoding the heavy and light chains ofmurine antibodies). The hybridoma cells serve as a preferred source ofsuch DNA. Once isolated, the DNA may be placed into expression vectors,which are then transfected into host-cells such as E. coli cells, simianCOS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that donot otherwise produce immunoglobulin protein, in order to synthesizemonoclonal antibodies in such recombinant host-cells. Review articles onrecombinant expression in bacteria of DNA encoding the antibody includeSkerra et al., Curr. Opin. Immunol., 5:256-262 (1993) and Plückthun,Immunol. Rev. 130:151-188 (1992).

In certain embodiments, anti-C1q, anti-C1s, anti-C1r, and/or anti-C1complex antibodies can be isolated from antibody phage librariesgenerated using the techniques described in McCafferty et al., Nature,348:552-554 (1990). Clackson et al., Nature, 352:624-628 (1991) andMarks et al., J. Mol. Biol., 222:581-597 (1991) described the isolationof murine and human antibodies, respectively, from phage libraries.Subsequent publications describe the production of high affinity(nanomolar (“nM”) range) human antibodies by chain shuffling (Marks etal., Bio/Technology, 10:779-783 (1992)), as well as combinatorialinfection and in vivo recombination as a strategy for constructing verylarge phage libraries (Waterhouse et al., Nucl. Acids Res., 21:2265-2266(1993)). Thus, these techniques are viable alternatives to traditionalmonoclonal antibody hybridoma techniques for isolation of monoclonalantibodies of desired specificity (e.g., those that bind a C1q, C1s, orC1r protein of the present disclosure).

The DNA encoding antibodies or fragments thereof may also be modified,for example, by substituting the coding sequence for human heavy- andlight-chain constant domains in place of the homologous murine sequences(U.S. Pat. No. 4,816,567; Morrison, et al., Proc. Natl Acad. Sci. USA,81:6851 (1984)), or by covalently joining to the immunoglobulin codingsequence all or part of the coding sequence for a non-immunoglobulinpolypeptide. Typically such non-immunoglobulin polypeptides aresubstituted for the constant domains of an antibody, or they aresubstituted for the variable domains of one antigen-combining site of anantibody to create a chimeric bivalent antibody comprising oneantigen-combining site having specificity for an antigen and anotherantigen-combining site having specificity for a different antigen.

The monoclonal antibodies described herein (e.g., anti-C1q, anti-C1s,anti-C1r, and/or anti-C1 complex antibodies of the present disclosure orfragments thereof) may by monovalent, the preparation of which is wellknown in the art. For example, one method involves recombinantexpression of immunoglobulin light chain and a modified heavy chain. Theheavy chain is truncated generally at any point in the Fc region so asto prevent heavy chain crosslinking. Alternatively, the relevantcysteine residues may be substituted with another amino acid residue orare deleted so as to prevent crosslinking. In vitro methods are alsosuitable for preparing monovalent antibodies. Digestion of antibodies toproduce fragments thereof, particularly Fab fragments, can beaccomplished using routine techniques known in the art.

Chimeric or hybrid anti-C1q, anti-C1s, anti-C1r, and/or anti-C1 complexantibodies also may be prepared in vitro using known methods insynthetic protein chemistry, including those involving crosslinkingagents. For example, immunotoxins may be constructed using adisulfide-exchange reaction or by forming a thioether bond. Examples ofsuitable reagents for this purpose include iminothiolate andmethyl-4-mercaptobutyrimidate.

(3) Humanized Antibodies

Anti-C1q, anti-C1s, anti-C1r, and/or anti-C1 complex antibodies of thepresent disclosure or antibody fragments thereof may further includehumanized or human antibodies. Humanized forms of non-human (e.g.,murine) antibodies are chimeric immunoglobulins, immunoglobulin chainsor fragments thereof (such as Fab, Fab′-SH, Fv, scFv, F(ab′)₂ or otherantigen-binding subsequences of antibodies) which contain minimalsequence derived from non-human immunoglobulin. Humanized antibodiesinclude human immunoglobulins (recipient antibody) in which residuesfrom a complementarity determining region (CDR) of the recipient arereplaced by residues from a CDR of a non-human species (donor antibody)such as mouse, rat or rabbit having the desired specificity, affinityand capacity. In some instances, Fv framework residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally will also comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. Jones et al., Nature 321: 522-525 (1986); Riechmann etal., Nature 332: 323-329 (1988) and Presta, Curr. Opin. Struct. Biol. 2:593-596 (1992).

Methods for humanizing non-human anti-C1q, anti-C1s, anti-C1r, and/oranti-C1 complex antibodies are well known in the art. Generally, ahumanized antibody has one or more amino acid residues introduced intoit from a source which is non-human. These non-human amino acid residuesare often referred to as “import” residues, which are typically takenfrom an “import” variable domain. Humanization can be essentiallyperformed following the method of Winter and co-workers, Jones et al.,Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988);Verhoeyen et al., Science 239:1534-1536 (1988), or through substitutingrodent CDRs or CDR sequences for the corresponding sequences of a humanantibody. Accordingly, such “humanized” antibodies are chimericantibodies (U.S. Pat. No. 4,816,567), wherein substantially less than anintact human variable domain has been substituted by the correspondingsequence from a non-human species. In practice, humanized antibodies aretypically human antibodies in which some CDR residues and possibly someFR residues are substituted by residues from analogous sites in rodentantibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important to reduceantigenicity. According to the so-called “best-fit” method, the sequenceof the variable domain of a rodent antibody is screened against theentire library of known human variable-domain sequences. The humansequence which is closest to that of the rodent is then accepted as thehuman framework (FR) for the humanized antibody. Sims et al., J.Immunol., 151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901(1987). Another method uses a particular framework derived from theconsensus sequence of all human antibodies of a particular subgroup oflight or heavy chains. The same framework may be used for severaldifferent humanized antibodies. Carter et al., Proc. Nat'l Acad. Sci.USA 89:4285 (1992); Presta et al., J. Immunol. 151:2623 (1993).

Furthermore, it is important that antibodies be humanized with retentionof high affinity for the antigen and other favorable biologicalproperties. To achieve this goal, according to a preferred method,humanized antibodies are prepared by a process of analyzing the parentalsequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the recipient and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen or antigens (e.g., C1q, C1s, or C1r proteins of thepresent disclosure), is achieved. In general, the CDR residues aredirectly and most substantially involved in influencing antigen binding.

Various forms of the humanized anti-C1q, anti-C1s, anti-C1r, and/oranti-C1 complex antibody are contemplated. For example, the humanizedanti-C1q, anti-C1s, anti-C1r, and/or anti-C1 complex antibody may be anantibody fragment, such as an Fab, which is optionally conjugated withone or more cytotoxic agent(s) in order to generate an immunoconjugate.Alternatively, the humanized anti-C1q, anti-C1s, anti-C1r, and/oranti-C1 complex antibody may be an intact antibody, such as an intactIgG1 antibody.

(4) Human Antibodies

Alternatively, human anti-C1q, anti-C1s, anti-C1r, and/or anti-C1complex antibodies can be generated. For example, it is now possible toproduce transgenic animals (e.g., mice) that are capable, uponimmunization, of producing a full repertoire of human antibodies in theabsence of endogenous immunoglobulin production. The homozygous deletionof the antibody heavy-chain joining region (J_(H)) gene in chimeric andgerm-line mutant mice results in complete inhibition of endogenousantibody production. Transfer of the human germ-line immunoglobulin genearray in such germ-line mutant mice will result in the production ofhuman antibodies upon antigen challenge. See, e.g., Jakobovits et al.,Proc. Nat'l Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature,362:255-258 (1993); Bruggermann et al., Year in Immunol., 7:33 (1993);U.S. Pat. No. 5,591,669 and WO 97/17852.

Alternatively, phage display technology can be used to produce humananti-C1q, anti-C1s, anti-C1r, and/or anti-C1 complex antibodies andantibody fragments in vitro, from immunoglobulin variable (V) domaingene repertoires from unimmunized donors. McCafferty et al., Nature348:552-553 (1990); Hoogenboom and Winter, J. Mol. Biol. 227: 381(1991). According to this technique, antibody V domain genes are clonedin-frame into either a major or minor coat protein gene of a filamentousbacteriophage, such as M13 or fd, and displayed as functional antibodyfragments on the surface of the phage particle. Because the filamentousparticle contains a single-stranded DNA copy of the phage genome,selections based on the functional properties of the antibody alsoresult in selection of the gene encoding the antibody exhibiting thoseproperties. Thus, the phage mimics some of the properties of the B-cell.Phage display can be performed in a variety of formats, reviewed in,e.g., Johnson, Kevin S. and Chiswell, David J., Curr. Opin Struct. Biol.3:564-571 (1993). Several sources of V-gene segments can be used forphage display. Clackson et al., Nature 352:624-628 (1991) isolated adiverse array of anti-oxazolone antibodies from a small randomcombinatorial library of V genes derived from the spleens of immunizedmice. A repertoire of V genes from unimmunized human donors can beconstructed and antibodies to a diverse array of antigens (includingself-antigens) can be isolated essentially following the techniquesdescribed by Marks et al., J. Mol. Biol. 222:581-597 (1991), or Griffithet al., EMBO J. 12:725-734 (1993). See also U.S. Pat. Nos. 5,565,332 and5,573,905. Additionally, yeast display technology can be used to producehuman anti-C1q, anti-C1s, anti-C1r, and/or anti-C1 complex antibodiesand antibody fragments in vitro (e.g., WO 2009/036379; WO 2010/105256;WO 2012/009568; US 2009/0181855; US 2010/0056386; and Feldhaus andSiegel (2004) J. Immunological Methods 290:69-80). In other embodiments,ribosome display technology can be used to produce human anti-C1q,anti-C1s, anti-C1r, and/or anti-C1 complex antibodies and antibodyfragments in vitro (e.g., Roberts and Szostak (1997) Proc Natl Acad Sci94:12297-12302; Schaffitzel et al. (1999) J. Immunolical Methods231:119-135; Lipovsek and Plückthun (2004) J. Immunological Methods290:51-67).

The techniques of Cole et al., and Boerner et al., are also availablefor the preparation of human anti-C1q, anti-C1s, anti-C1r, and/oranti-C1 complex monoclonal antibodies (Cole et al., MonoclonalAntibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner etal., J. Immunol. 147(1): 86-95 (1991). Similarly, human anti-C1q,anti-C1s, anti-C1r, and/or anti-C1 complex antibodies can be made byintroducing human immunoglobulin loci into transgenic animals, e.g.,mice in which the endogenous immunoglobulin genes have been partially orcompletely inactivated. Upon challenge, human antibody production isobserved, which closely resembles that seen in humans in all respects,including gene rearrangement, assembly and antibody repertoire. Thisapproach is described, for example, in U.S. Pat. Nos. 5,545,807;5,545,806, 5,569,825, 5,625,126, 5,633,425, 5,661,016 and in thefollowing scientific publications: Marks et al., Bio/Technology 10:779-783 (1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison,Nature 368: 812-13 (1994), Fishwild et al., Nature Biotechnology 14:845-51 (1996), Neuberger, Nature Biotechnology 14: 826 (1996) andLonberg and Huszar, Intern. Rev. Immunol. 13: 65-93 (1995).

Finally, human anti-C1q, anti-C1s, anti-C1r, and/or anti-C1 complexantibodies may also be generated in vitro by activated B-cells (see U.S.Pat. Nos. 5,567,610 and 5,229,275).

(5) Antibody Fragments

In certain embodiments there are advantages to using anti-C1q, anti-C1s,anti-C1r, and/or anti-C1 complex antibody fragments, rather than wholeanti-C1q, anti-C1s, anti-C1r, and/or anti-C1 complex antibodies. Smallerfragment sizes allow for rapid clearance.

Various techniques have been developed for the production of antibodyfragments. Traditionally, these fragments were derived via proteolyticdigestion of intact antibodies (see, e.g., Morimoto et al., J. Biochem.Biophys. Method. 24:107-117 (1992); and Brennan et al., Science 229:81(1985)). However, these fragments can now be produced directly byrecombinant host-cells, for example, using nucleic acids encodinganti-C1q, anti-C1s, anti-C1r, and/or anti-C1 complex antibodies of thepresent disclosure. Fab, Fv and scFv antibody fragments can all beexpressed in and secreted from E. coli, thus allowing thestraightforward production of large amounts of these fragments.Anti-C1q, anti-C1s, anti-C1r, and/or anti-C1 complex antibody fragmentscan also be isolated from the antibody phage libraries as discussedabove. Alternatively, Fab′-SH fragments can be directly recovered fromE. coli and chemically coupled to form F(ab′)₂ fragments (Carter et al.,Bio/Technology 10:163-167 (1992)). According to another approach,F(ab′)₂ fragments can be isolated directly from recombinant host-cellculture. Production of Fab and F(ab′)₂ antibody fragments with increasedin vivo half-lives are described in U.S. Pat. No. 5,869,046. In otherembodiments, the antibody of choice is a single chain Fv fragment(scFv). See WO 93/16185; U.S. Pat. No. 5,571,894 and U.S. Pat. No.5,587,458. The anti-C1q, anti-C1s, anti-C1r, and/or anti-C1 complexantibody fragment may also be a “linear antibody,” e.g., as described inU.S. Pat. No. 5,641,870. Such linear antibody fragments may bemonospecific or bispecific.

(6) Bispecific and Polyspecific Antibodies

Bispecific antibodies (BsAbs) are antibodies that have bindingspecificities for at least two different epitopes, including those onthe same or another protein (e.g., one or more C1q, C1s, or C1r proteinsof the present disclosure). Alternatively, one part of a BsAb can bearmed to bind to the target C1q, C1s, or C1r antigen, and another can becombined with an arm that binds to a second protein. Such antibodies canbe derived from full length antibodies or antibody fragments (e.g.,F(ab′)₂ bispecific antibodies).

Methods for making bispecific antibodies are known in the art.Traditional production of full length bispecific antibodies is based onthe coexpression of two immunoglobulin heavy-chain/light chain pairs,where the two chains have different specificities. Millstein et al.,Nature, 305:537-539 (1983). Because of the random assortment ofimmunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of 10 different antibody molecules, of whichonly one has the correct bispecific structure. Purification of thecorrect molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed in WO 93/08829 and in Traunecker et al., EMBOJ., 10:3655-3659 (1991).

According to a different approach, antibody variable domains with thedesired binding specificities (antibody-antigen combining sites) arefused to immunoglobulin constant domain sequences. The fusion preferablyis with an immunoglobulin heavy chain constant domain, comprising atleast part of the hinge, C_(H)2, and C_(H)3 regions. It is preferred tohave the first heavy-chain constant region (C_(H)1) containing the sitenecessary for light chain binding, present in at least one of thefusions. DNAs encoding the immunoglobulin heavy chain fusions and, ifdesired, the immunoglobulin light chain, are inserted into separateexpression vectors, and are co-transfected into a suitable hostorganism. This provides for great flexibility in adjusting the mutualproportions of the three polypeptide fragments in embodiments whenunequal ratios of the three polypeptide chains used in the constructionprovide the optimum yields. It is, however, possible to insert thecoding sequences for two or all three polypeptide chains in oneexpression vector when the expression of at least two polypeptide chainsin equal ratios results in high yields or when the ratios are of noparticular significance.

In a preferred embodiment of this approach, the bispecific antibodiesare composed of a hybrid immunoglobulin heavy chain with a first bindingspecificity in one arm, and a hybrid immunoglobulin heavy chain-lightchain pair (providing a second binding specificity) in the other arm. Itwas found that this asymmetric structure facilitates the separation ofthe desired bispecific compound from unwanted immunoglobulin chaincombinations, as the presence of an immunoglobulin light chain in onlyhalf of the bispecific molecules provides for an easy way of separation.This approach is disclosed in WO 94/04690. For further details ofgenerating bispecific antibodies, see, for example, Suresh et al.,Methods in Enzymology 121: 210 (1986).

According to another approach described in WO 96/27011 or U.S. Pat. No.5,731,168, the interface between a pair of antibody molecules can beengineered to maximize the percentage of heterodimers which arerecovered from recombinant-cell culture. The preferred interfacecomprises at least a part of the C_(H)3 region of an antibody constantdomain. In this method, one or more small amino acid side chains fromthe interface of the first antibody molecule are replaced with largerside chains (e.g., tyrosine or tryptophan). Compensatory “cavities” ofidentical or similar size to the large side chains(s) are created on theinterface of the second antibody molecule by replacing large amino acidside chains with smaller ones (e.g., alanine or threonine). Thisprovides a mechanism for increasing the yield of the heterodimer overother unwanted end-products such as homodimers.

Techniques for generating bispecific antibodies from antibody fragmentshave been described in the literature. For example, bispecificantibodies can be prepared using chemical linkage. Brennan et al.,Science 229:81 (1985) describe a procedure wherein intact antibodies areproteolytically cleaved to generate F(ab′)₂ fragments. These fragmentsare reduced in the presence of the dithiol complexing agent sodiumarsenite to stabilize vicinal dithiols and prevent intermoleculardisulfide formation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-TNB derivative to form the bispecificantibody. The bispecific antibodies produced can be used as agents forthe selective immobilization of enzymes.

Fab′ fragments may be directly recovered from E. coli and chemicallycoupled to form bispecific antibodies. Shalaby et al., J. Exp. Med. 175:217-225 (1992) describes the production of fully humanized bispecificantibody F(ab′)₂ molecules. Each Fab′ fragment was separately secretedfrom E. coli and subjected to directed chemical coupling in vitro toform the bispecific antibody. The bispecific antibody thus formed wasable to bind to cells overexpressing the ErbB2 receptor and normal humanT-cells, as well as trigger the lytic activity of human cytotoxiclymphocytes against human breast tumor targets.

Various techniques for making and isolating bivalent antibody fragmentsdirectly from recombinant-cell culture have also been described. Forexample, bivalent heterodimers have been produced using leucine zippers.Kostelny et al., J. Immunol., 148(5):1547-1553 (1992). The leucinezipper peptides from the Fos and Jun proteins were linked to the Fab′portions of two different antibodies by gene fusion. The antibodyhomodimers were reduced at the hinge region to form monomers and thenre-oxidized to form the antibody heterodimers. The “diabody” technologydescribed by Hollinger et al., Proc. Nat'l Acad. Sci. USA, 90: 6444-6448(1993) has provided an alternative mechanism for makingbispecific/bivalent antibody fragments. The fragments comprise aheavy-chain variable domain (V_(H)) connected to a light-chain variabledomain (V_(L)) by a linker which is too short to allow pairing betweenthe two domains on the same chain. Accordingly, the V_(H) and V_(L)domains of one fragment are forced to pair with the complementary V_(L)and V_(H) domains of another fragment, thereby forming twoantigen-binding sites. Another strategy for making bispecific/bivalentantibody fragments by the use of single-chain Fv (sFv) dimers has alsobeen reported. See Gruber et al., J. Immunol., 152:5368 (1994).

Antibodies with more than two valencies are also contemplated. Forexample, trispecific antibodies can be prepared. Tutt et al., J.Immunol. 147:60 (1991).

Exemplary bispecific antibodies may bind to two different antigens. Insome embodiments a bispecific antibody binds to a first antigen C1q,C1r, or C1s and a second antigen facilitating transport across theblood-brain barrier. Numerous antigens are known in the art thatfacilitate transport across the blood-brain barrier (see, e.g.,Gabathuler R., Approaches to transport therapeutic drugs across theblood-brain barrier to treat brain diseases, Neurobiol. Dis. 37 (2010)48-57). Such second antigens include, without limitation, transferrinreceptor (TR), insulin receptor (HIR), Insulin-like growth factorreceptor (IGFR), low-density lipoprotein receptor related proteins 1 and2 (LPR-1 and 2), diphtheria toxin receptor, including CRM197 (anon-toxic mutant of diphtheria toxin), llama single domain antibodiessuch as TMEM 30(A) (Flippase), protein transduction domains such as TAT,Syn-B, or penetratin, poly-arginine or generally positively chargedpeptides, and Angiopep peptides such as ANG1005 (see, e.g., Gabathuler,2010).

(7) Multivalent Antibodies

A multivalent antibody may be internalized (and/or catabolized) fasterthan a bivalent antibody by a cell expressing an antigen to which theantibodies bind. The anti-C1q, anti-C1s, anti-C1r, and/or anti-C1complex antibodies of the present disclosure or antibody fragmentsthereof can be multivalent antibodies (which are other than of the IgMclass) with three or more antigen binding sites (e.g., tetravalentantibodies), which can be readily produced by recombinant expression ofnucleic acid encoding the polypeptide chains of the antibody. Themultivalent antibody can comprise a dimerization domain and three ormore antigen binding sites. The preferred dimerization domain comprisesan Fc region or a hinge region. In this scenario, the antibody willcomprise an Fc region and three or more antigen binding sitesamino-terminal to the Fc region. The preferred multivalent antibodyherein contains three to about eight, but preferably four, antigenbinding sites. The multivalent antibody contains at least onepolypeptide chain (and preferably two polypeptide chains), wherein thepolypeptide chain or chains comprise two or more variable domains. Forinstance, the polypeptide chain or chains may compriseVD1-(X1)n-VD2-(X2)n-Fc, wherein VD1 is a first variable domain, VD2 is asecond variable domain, Fc is one polypeptide chain of an Fc region, X1and X2 represent an amino acid or polypeptide, and n is 0 or 1.Similarly, the polypeptide chain or chains may compriseV_(H)-C_(H)1-flexible linker-V_(H)-C_(H)1-Fc region chain; orV_(H)-C_(H)1-V_(H)-C_(H)1-Fc region chain. The multivalent antibodyherein preferably further comprises at least two (and preferably four)light chain variable domain polypeptides. The multivalent antibodyherein may, for instance, comprise from about two to about eight lightchain variable domain polypeptides. The light chain variable domainpolypeptides contemplated here comprise a light chain variable domainand, optionally, further comprise a CL domain.

(8) Heteroconjugate Antibodies

Heteroconjugate antibodies are also within the scope of the presentdisclosure.

Heteroconjugate antibodies are composed of two covalently joinedantibodies (e.g., anti-C1q, anti-C1s, anti-C1r, and/or anti-C1 complexantibodies of the present disclosure or antibody fragments thereof). Forexample, one of the antibodies in the heteroconjugate can be coupled toavidin, the other to biotin. Such antibodies have, for example, beenproposed to target immune system cells to unwanted cells, U.S. Pat. No.4,676,980, and have been used to treat HIV infection. InternationalPublication Nos. WO 91/00360, WO 92/200373 and EP 0308936. It iscontemplated that the antibodies may be prepared in vitro using knownmethods in synthetic protein chemistry, including those involvingcrosslinking agents. For example, immunotoxins may be constructed usinga disulfide exchange reaction or by forming a thioether bond. Examplesof suitable reagents for this purpose include iminothiolate andmethyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S.Pat. No. 4,676,980. Heteroconjugate antibodies may be made using anyconvenient cross-linking methods. Suitable cross-linking agents are wellknown in the art, and are disclosed in U.S. Pat. No. 4,676,980, alongwith a number of cross-linking techniques.

(9) Effector Function Engineering

It may also be desirable to modify an anti-C1q, anti-C1s, anti-C1r,and/or anti-C1 complex antibody of the present disclosure to modifyeffector function and/or to increase serum half-life of the antibody.For example, the Fc receptor binding site on the constant region may bemodified or mutated to remove or reduce binding affinity to certain Fcreceptors, such as FcγRI, FcγRII, and/or FcγRIII. In some embodiments,the effector function is impaired by removing N-glycosylation of the Fcregion (e.g., in the CH 2 domain of IgG) of the antibody. In someembodiments, the effector function is impaired by modifying regions suchas 233-236, 297, and/or 327-331 of human IgG as described in PCT WO99/58572 and Armour et al., Molecular Immunology 40: 585-593 (2003);Reddy et al., J. Immunology 164:1925-1933 (2000).

The constant region of the anti-complement antibodies described hereinmay also be modified to impair complement activation. For example,complement activation of IgG antibodies following binding of the C1component of complement may be reduced by mutating amino acid residuesin the constant region in a C1 binding motif (e.g., C1q binding motif).It has been reported that Ala mutation for each of D270, K322, P329,P331 of human IgG1 significantly reduced the ability of the antibody tobind to C1q and activating complement. For murine IgG2b, C1s bindingmotif constitutes residues E318, K320, and K322. Idusogie et al. (2000)J. Immunology 164:4178-4184; Duncan et al. (1988) Nature 322: 738-740.As the C1q binding motif E318, K320, and K322 identified for murineIgG2b is believed to be common for other antibody isotypes (Duncan etal. (1988) Nature 322:738-740), C1q, C1s, C1r, or C1 complex bindingactivity for IgG2b can be abolished by replacing any one of the threespecified residues with a residue having an inappropriate functionalityon its side chain. It is not necessary to replace the ionic residuesonly with Ala to abolish C1q, C1s, C1r, or C1 complex binding. It isalso possible to use other alkyl-substituted non-ionic residues, such asGly, Ile, Leu, or Val, or such aromatic non-polar residues as Phe, Tyr,Trp and Pro in place of any one of the three residues in order toabolish C1q, C1s, C1r, or C1 complex binding. In addition, it is alsopossible to use such polar non-ionic residues as Ser, Thr, Cys, and Metin place of residues 320 and 322, but not 318, in order to abolish C1q,C1s, C1r, or C1 complex binding activity. In addition, removal ofcarbohydrate modifications of the Fc region necessary for complementbinding can prevent complement activation Glycosylation of a conservedasparagine (Asn-297) on the CH2 domain of IgG heavy chains is essentialfor antibody effector functions (Jefferis et al. (1998) Immunol Rev163:59-76). Modification of the Fc glycan alters IgG conformation andreduces the Fc affinity for binding of complement protein C1q, C1s, orC1r and effector cell receptor FcR (Alhorn et al. (2008) PLos ONE 2008;3:e1413). Complete removal of the Fc glycan abolishes CDC and ADCC.Deglycosylation can be performed using glycosidase enzymes for exampleEndoglycosidase S (EndoS), a 108 kDa enzyme encoded by the gene endoS ofStreptococcus pyogenes that selectively digests asparagine-linkedglycans on the heavy chain of all IgG subclasses, without action onother immunoglobulin classes or other glycoproteins (Collin et al.(2001) EMBO J 2001; 20:3046-3055).

To increase the serum half-life of the antibody, one may incorporate asalvage receptor binding epitope into the antibody (especially anantibody fragment) as described in U.S. Pat. No. 5,739,277, for example.As used herein, the term “salvage receptor binding epitope” refers to anepitope of the Fc region of an IgG molecule (e.g., IgG₁, IgG₂, IgG₃, orIgG₄) that is responsible for increasing the in vivo serum half-life ofthe IgG molecule.

(10) Other Amino Acid Sequence Modifications

Amino acid sequence modifications of anti-C1q, anti-C1s, anti-C1r,and/or anti-C1 complex antibodies of the present disclosure, or antibodyfragments thereof, are also contemplated. For example, it may bedesirable to improve the binding affinity and/or other biologicalproperties of the antibodies or antibody fragments. Amino acid sequencevariants of the antibodies or antibody fragments are prepared byintroducing appropriate nucleotide changes into the nucleic acidencoding the antibodies or antibody fragments, or by peptide synthesis.Such modifications include, for example, deletions from, and/orinsertions into and/or substitutions of, residues within the amino acidsequences of the antibody. Any combination of deletion, insertion, andsubstitution is made to arrive at the final construct, provided that thefinal construct possesses the desired characteristics (i.e., the abilityto bind or physically interact with a C1q, C1s, or C1r protein of thepresent disclosure). The amino acid changes also may alterpost-translational processes of the antibody, such as changing thenumber or position of glycosylation sites.

A useful method for identification of certain residues or regions of theanti-C1q, anti-C1s, anti-C1r, and/or anti-C1 complex antibody that arepreferred locations for mutagenesis is called “alanine scanningmutagenesis” as described by Cunningham and Wells in Science,244:1081-1085 (1989). Here, a residue or group of target residues areidentified (e.g., charged residues such as arg, asp, his, lys, and glu)and replaced by a neutral or negatively charged amino acid (mostpreferably alanine or polyalanine) to affect the interaction of theamino acids with the target antigen. Those amino acid locationsdemonstrating functional sensitivity to the substitutions then arerefined by introducing further or other variants at, or for, the sitesof substitution. Thus, while the site for introducing an amino acidsequence variation is predetermined, the nature of the mutation per seneed not be predetermined. For example, to analyze the performance of amutation at a given site, alanine scanning or random mutagenesis isconducted at the target codon or region and the expressed antibodyvariants are screened for the desired activity.

Amino acid sequence insertions include amino-(“N”) and/or carboxy-(“C”)terminal fusions ranging in length from one residue to polypeptidescontaining a hundred or more residues, as well as intrasequenceinsertions of single or multiple amino acid residues. Examples ofterminal insertions include an antibody with an N-terminal methionylresidue or the antibody fused to a cytotoxic polypeptide. Otherinsertional variants of the antibody molecule include the fusion to theN- or C-terminus of the antibody to an enzyme or a polypeptide whichincreases the serum half-life of the antibody.

Another type of variant is an amino acid substitution variant. Thesevariants have at least one amino acid residue in the antibody moleculereplaced by a different residue. The sites of greatest interest forsubstitutional mutagenesis include the hypervariable regions, but FRalterations are also contemplated. Conservative substitutions are shownin the Table A below under the heading of “preferred substitutions”. Ifsuch substitutions result in a change in biological activity, then moresubstantial changes, denominated “exemplary substitutions” in Table A,or as further described below in reference to amino acid classes, may beintroduced and the products screened.

TABLE A Amino Acid Substitutions Original Exemplary Preferred ResidueSubstitutions Substitutions Ala (A) val; leu; ile val Arg (R) lys; gln;asn lys Asn (N) gln; his; asp, lys; arg gln Asp (D) glu; asn glu Cys (C)ser; ala ser Gln (Q) asn; glu asn Glu (E) asp; gln asp Gly (G) ala alaHis (H) asn; gln; lys; arg arg Ile (I) leu; val; met; ala; phe;norleucine leu Leu (L) norleucine; ile; val; met; ala; phe ile Lys (K)arg; gln; asn arg Met (M) leu; phe; ile leu Phe (F) leu; val; ile; ala;tyr tyr Pro (P) ala ala Ser (S) thr thr Thr (T) ser ser Trp (W) tyr; phetyr Tyr (Y) trp; phe; thr; ser phe Val (V) ile; leu; met; phe; ala;norleucine leu

Substantial modifications in the biological properties of the antibodyare accomplished by selecting substitutions that differ significantly intheir effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain. Naturallyoccurring residues are divided into groups based on common side-chainproperties:

(1) hydrophobic: norleucine, met, ala, val, leu, ile;

(2) neutral hydrophilic: cys, ser, thr;

(3) acidic: asp, glu;

(4) basic: asn, gln, his, lys, arg;

(5) residues that influence chain orientation: gly, pro; and

(6) aromatic: trp, tyr, phe.

Non-conservative substitutions entail exchanging a member of one ofthese classes for another class.

Any cysteine residue not involved in maintaining the proper conformationof the antibody also may be substituted, generally with serine, toimprove the oxidative stability of the molecule and prevent aberrantcrosslinking Conversely, cysteine bond(s) may be added to the antibodyto improve its stability (particularly where the antibody is an antibodyfragment, such as an Fv fragment).

A particularly preferred type of substitutional variant involvessubstituting one or more hypervariable region residues of a parentantibody (e.g. a humanized or human anti-C1q, anti-C1s, anti-C1r, and/oranti-C1 complex antibody). Generally, the resulting variant(s) selectedfor further development will have improved biological propertiesrelative to the parent antibody from which they are generated. Aconvenient way for generating such substitutional variants involvesaffinity maturation using phage display. Briefly, several hypervariableregion sites (e.g., 6-7 sites) are mutated to generate all possibleamino substitutions at each site. The antibody variants thus generatedare displayed in a monovalent fashion from filamentous phage particlesas fusions to the gene III product of M13 packaged within each particle.The phage-displayed variants are then screened for their biologicalactivity (e.g., binding affinity) as herein disclosed. In order toidentify candidate hypervariable region sites for modification, alaninescanning mutagenesis can be performed to identify hypervariable regionresidues contributing significantly to antigen binding. Alternatively,or additionally, it may be beneficial to analyze a crystal structure ofthe antigen-antibody complex to identify contact points between theantibody and the antigen (e.g., a C1q, C1s, or C1r protein of thepresent disclosure). Such contact residues and neighboring residues arecandidates for substitution according to the techniques elaboratedherein. Once such variants are generated, the panel of variants issubjected to screening as described herein and antibodies with superiorproperties in one or more relevant assays may be selected for furtherdevelopment.

Another type of amino acid variant of the antibody alters the originalglycosylation pattern of the antibody. By altering is meant deleting oneor more carbohydrate moieties found in the antibody, and/or adding oneor more glycosylation sites that are not present in the antibody.

Glycosylation of antibodies is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to the antibody is convenientlyaccomplished by altering the amino acid sequence such that it containsone or more of the above-described tripeptide sequences (for N-linkedglycosylation sites). The alteration may also be made by the additionof, or substitution by, one or more serine or threonine residues to thesequence of the original antibody (for O-linked glycosylation sites).

Nucleic acid molecules encoding amino acid sequence variants of theanti-IgE antibody are prepared by a variety of methods known in the art.These methods include, but are not limited to, isolation from a naturalsource (in the case of naturally occurring amino acid sequence variants)or preparation by oligonucleotide-mediated (or site-directed)mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlierprepared variant or a non-variant version of the antibodies (e.g., ananti-C1q, anti-C1s, anti-C1r, and/or anti-C1 complex antibody of thepresent disclosure) or antibody fragments.

(11) Other Antibody Modifications

Anti-C1q, anti-C1s, anti-C1r, and/or anti-C1 complex antibodies of thepresent disclosure, or antibody fragments thereof, can be furthermodified to contain additional non-proteinaceous moieties that are knownin the art and readily available. Preferably, the moieties suitable forderivatization of the antibody are water-soluble polymers. Non-limitingexamples of water-soluble polymers include, but are not limited to,polyethylene glycol (PEG), copolymers of ethylene glycol/propyleneglycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1, 3-dioxolane, poly-1,3,6-trioxane, ethylene/maleicanhydride copolymer, polyaminoacids (either homopolymers or randomcopolymers), and dextran or poly(n-vinyl pyrrolidone)polyethyleneglycol, polypropylene glycol homopolymers, polypropylene oxide/ethyleneoxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinylalcohol, and mixtures thereof. Polyethylene glycol propionaldehyde mayhave advantages in manufacturing due to its stability in water. Thepolymer may be of any molecular weight, and may be branched orunbranched. The number of polymers attached to the antibody may vary,and if more than one polymer is attached, they can be the same ordifferent molecules. In general, the number and/or type of polymers usedfor derivatization can be determined based on considerations including,but not limited to, the particular properties or functions of theantibody to be improved, whether the antibody derivative will be used ina therapy under defined conditions, etc. Such techniques and othersuitable formulations are disclosed in Remington: The Science andPractice of Pharmacy, 20th Ed., Alfonso Gennaro, Ed., PhiladelphiaCollege of Pharmacy and Science (2000).

Nucleic Acids, Vectors, and Host Cells

Anti-C1q, anti-C1s, anti-C1r, and/or anti-C1 complex antibodies of thepresent disclosure may be produced using recombinant methods andcompositions, e.g., as described in U.S. Pat. No. 4,816,567. In someembodiments, isolated nucleic acids having a nucleotide sequenceencoding any of the anti-C1q, anti-C1s, anti-C1r, and/or anti-C1 complexantibodies of the present disclosure are provided. Such nucleic acidsmay encode an amino acid sequence containing the VL and/or an amino acidsequence containing the VH of the anti-C1q, anti-C1s, anti-C1r, and/oranti-C1 complex antibody (e.g., the light and/or heavy chains of theantibody). In some embodiments, one or more vectors (e.g., expressionvectors) containing such nucleic acids are provided. In someembodiments, a host cell containing such nucleic acid is also provided.In some embodiments, the host cell contains (e.g., has been transducedwith): (1) a vector containing a nucleic acid that encodes an amino acidsequence containing the VL of the antibody and an amino acid sequencecontaining the VH of the antibody, or (2) a first vector containing anucleic acid that encodes an amino acid sequence containing the VL ofthe antibody and a second vector containing a nucleic acid that encodesan amino acid sequence containing the VH of the antibody. In someembodiments, the host cell is eukaryotic, e.g., a Chinese Hamster Ovary(CHO) cell or lymphoid cell (e.g., Y0, NS0, Sp20 cell).

Methods of making an anti-C1q, anti-C1s, anti-C1r, and/or anti-C1complex antibody of the present disclosure are provided. In someembodiments, the method includes culturing a host cell of the presentdisclosure containing a nucleic acid encoding the anti-C1q, anti-C1s,anti-C1r, and/or anti-C1 complex antibody, under conditions suitable forexpression of the antibody. In some embodiments, the antibody issubsequently recovered from the host cell (or host cell culture medium).

For recombinant production of an anti-C1q, anti-C1s, anti-C1r, and/oranti-C1 complex antibody of the present disclosure, a nucleic acidencoding the anti-C1q, anti-C1s, anti-C1r, and/or anti-C1 complexantibody is isolated and inserted into one or more vectors for furthercloning and/or expression in a host cell. Such nucleic acid may bereadily isolated and sequenced using conventional procedures (e.g., byusing oligonucleotide probes that are capable of binding specifically togenes encoding the heavy and light chains of the antibody).

Suitable vectors containing a nucleic acid sequence encoding any of theanti-C1q, anti-C1s, anti-C1r, and/or anti-C1 complex antibodies of thepresent disclosure, or fragments thereof polypeptides (includingantibodies) described herein include, without limitation, cloningvectors and expression vectors. Suitable cloning vectors can beconstructed according to standard techniques, or may be selected from alarge number of cloning vectors available in the art. While the cloningvector selected may vary according to the host cell intended to be used,useful cloning vectors generally have the ability to self-replicate, maypossess a single target for a particular restriction endonuclease,and/or may carry genes for a marker that can be used in selecting clonescontaining the vector. Suitable examples include plasmids and bacterialviruses, e.g., pUC18, pUC19, Bluescript (e.g., pBS SK+) and itsderivatives, mpl8, mpl9, pBR322, pMB9, ColE1, pCR1, RP4, phage DNAs, andshuttle vectors such as pSA3 and pAT28. These and many other cloningvectors are available from commercial vendors such as BioRad,Strategene, and Invitrogen.

Expression vectors generally are replicable polynucleotide constructsthat contain a nucleic acid of the present disclosure. The expressionvector may replicable in the host cells either as episomes or as anintegral part of the chromosomal DNA. Suitable expression vectorsinclude but are not limited to plasmids, viral vectors, includingadenoviruses, adeno-associated viruses, retroviruses, cosmids, andexpression vector(s) disclosed in PCT Publication No. WO 87/04462.Vector components may generally include, but are not limited to, one ormore of the following: a signal sequence; an origin of replication; oneor more marker genes; suitable transcriptional controlling elements(such as promoters, enhancers and terminator). For expression (i.e.,translation), one or more translational controlling elements are alsousually required, such as ribosome binding sites, translation initiationsites, and stop codons.

The vectors containing the nucleic acids of interest can be introducedinto the host cell by any of a number of appropriate means, includingelectroporation, transfection employing calcium chloride, rubidiumchloride, calcium phosphate, DEAE-dextran, or other substances;microprojectile bombardment; lipofection; and infection (e.g., where thevector is an infectious agent such as vaccinia virus). The choice ofintroducing vectors or polynucleotides will often depend on features ofthe host cell. In some embodiments, the vector contains a nucleic acidcontaining one or more amino acid sequences encoding an anti-C1q,anti-C1s, anti-C1r, and/or anti-C1 complex antibody of the presentdisclosure.

Suitable host cells for cloning or expression of antibody-encodingvectors include prokaryotic or eukaryotic cells. For example, anti-C1q,anti-C1s, anti-C1r, and/or anti-C1 complex antibodies of the presentdisclosure may be produced in bacteria, in particular when glycosylationand Fc effector function are not needed. For expression of antibodyfragments and polypeptides in bacteria (e.g., U.S. Pat. Nos. 5,648,237,5,789,199, and 5,840,523; and Charlton, Methods in Molecular Biology,Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, N.J., 2003), pp.245-254, describing expression of antibody fragments in E. coli.). Afterexpression, the antibody may be isolated from the bacterial cell pastein a soluble fraction and can be further purified.

In addition to prokaryotes, eukaryotic microorganisms, such asfilamentous fungi or yeast, are also suitable cloning or expressionhosts for antibody-encoding vectors, including fungi and yeast strainswhose glycosylation pathways have been “humanized,” resulting in theproduction of an antibody with a partially or fully human glycosylationpattern (e.g., Gerngross, Nat. Biotech. 22:1409-1414 (2004); and Li etal., Nat. Biotech. 24:210-215 (2006)).

Suitable host cells for the expression of glycosylated antibody can alsobe derived from multicellular organisms (invertebrates and vertebrates).Examples of invertebrate cells include plant and insect cells. Numerousbaculoviral strains have been identified which may be used inconjunction with insect cells, particularly for transfection ofSpodoptera frugiperda cells. Plant cell cultures can also be utilized ashosts (e.g., U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978,and 6,417,429, describing PLANTIBODIES™ technology for producingantibodies in transgenic plants.).

Vertebrate cells may also be used as hosts. For example, mammalian celllines that are adapted to grow in suspension may be useful. Otherexamples of useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-7); human embryonic kidney line (293 or 293cells as described, e.g., in Graham et al., J. Gen Virol. 36:59 (1977));baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells asdescribed, e.g., in Mather, Biol. Reprod. 23:243-251 (1980)); monkeykidney cells (CV1); African green monkey kidney cells (VERO-76); humancervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo ratliver cells (BRL 3A); human lung cells (W138); human liver cells (HepG2); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g., inMather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5 cells; andFS4 cells. Other useful mammalian host cell lines include Chinesehamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al.,Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines suchas Y0, NS0 and Sp2/0. For a review of certain mammalian host cell linessuitable for antibody production, see, e.g., Yazaki and Wu, Methods inMolecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa,N.J.), pp. 255-268 (2003).

Pharmaceutical Compositions

Anti-C1q, anti-C1s, anti-C1r, and/or anti-C1 complex antibodies of thepresent disclosure can be incorporated into a variety of formulationsfor therapeutic use (e.g., by administration) or in the manufacture of amedicament (e.g., for treating or preventing an autoimmune orneurodegenerative disease) by combining the antibodies with appropriatepharmaceutically acceptable carriers or diluents, and may be formulatedinto preparations in solid, semi-solid, liquid or gaseous forms.Examples of such formulations include, without limitation, tablets,capsules, powders, granules, ointments, solutions, suppositories,injections, inhalants, gels, microspheres, and aerosols. Pharmaceuticalcompositions can include, depending on the formulation desired,pharmaceutically-acceptable, non-toxic carriers of diluents, which arevehicles commonly used to formulate pharmaceutical compositions foranimal or human administration. The diluent is selected so as not toaffect the biological activity of the combination. Examples of suchdiluents include, without limitation, distilled water, buffered water,physiological saline, PBS, Ringer's solution, dextrose solution, andHank's solution. A pharmaceutical composition or formulation of thepresent disclosure can further include other carriers, adjuvants, ornon-toxic, nontherapeutic, nonimmunogenic stabilizers, excipients andthe like. The compositions can also include additional substances toapproximate physiological conditions, such as pH adjusting and bufferingagents, toxicity adjusting agents, wetting agents and detergents.

A pharmaceutical composition of the present disclosure can also includeany of a variety of stabilizing agents, such as an antioxidant forexample. When the pharmaceutical composition includes a polypeptide, thepolypeptide can be complexed with various well-known compounds thatenhance the in vivo stability of the polypeptide, or otherwise enhanceits pharmacological properties (e.g., increase the half-life of thepolypeptide, reduce its toxicity, and enhance solubility or uptake).Examples of such modifications or complexing agents include, withoutlimitation, sulfate, gluconate, citrate and phosphate. The polypeptidesof a composition can also be complexed with molecules that enhance theirin vivo attributes. Such molecules include, without limitation,carbohydrates, polyamines, amino acids, other peptides, ions (e.g.,sodium, potassium, calcium, magnesium, manganese), and lipids.

Further examples of formulations that are suitable for various types ofadministration can be found in Remington's Pharmaceutical Sciences, MacePublishing Company, Philadelphia, Pa., 17th ed. (1985). For a briefreview of methods for drug delivery, see, Langer, Science 249:1527-1533(1990).

For oral administration, the active ingredient can be administered insolid dosage forms, such as capsules, tablets, and powders, or in liquiddosage forms, such as elixirs, syrups, and suspensions. The activecomponent(s) can be encapsulated in gelatin capsules together withinactive ingredients and powdered carriers, such as glucose, lactose,sucrose, mannitol, starch, cellulose or cellulose derivatives, magnesiumstearate, stearic acid, sodium saccharin, talcum, magnesium carbonate.Examples of additional inactive ingredients that may be added to providedesirable color, taste, stability, buffering capacity, dispersion orother known desirable features are red iron oxide, silica gel, sodiumlauryl sulfate, titanium dioxide, and edible white ink. Similar diluentscan be used to make compressed tablets. Both tablets and capsules can bemanufactured as sustained release products to provide for continuousrelease of medication over a period of hours. Compressed tablets can besugar coated or film coated to mask any unpleasant taste and protect thetablet from the atmosphere, or enteric-coated for selectivedisintegration in the gastrointestinal tract. Liquid dosage forms fororal administration can contain coloring and flavoring to increasepatient acceptance.

Formulations suitable for parenteral administration include aqueous andnon-aqueous, isotonic sterile injection solutions, which can containantioxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.

The components used to formulate the pharmaceutical compositions arepreferably of high purity and are substantially free of potentiallyharmful contaminants (e.g., at least National Food (NF) grade, generallyat least analytical grade, and more typically at least pharmaceuticalgrade). Moreover, compositions intended for in vivo use are usuallysterile. To the extent that a given compound must be synthesized priorto use, the resulting product is typically substantially free of anypotentially toxic agents, particularly any endotoxins, which may bepresent during the synthesis or purification process. Compositions forparental administration are also sterile, substantially isotonic andmade under GMP conditions.

Formulations may be optimized for retention and stabilization in thebrain or central nervous system. When the agent is administered into thecranial compartment, it is desirable for the agent to be retained in thecompartment, and not to diffuse or otherwise cross the blood brainbarrier. Stabilization techniques include cross-linking, multimerizing,or linking to groups such as polyethylene glycol, polyacrylamide,neutral protein carriers, etc. in order to achieve an increase inmolecular weight.

Other strategies for increasing retention include the entrapment of theantibody, such as an anti-C1q, anti-C1s, anti-C1r, and/or anti-C1complex antibody of the present disclosure, in a biodegradable orbioerodible implant. The rate of release of the therapeutically activeagent is controlled by the rate of transport through the polymericmatrix, and the biodegradation of the implant. The transport of drugthrough the polymer barrier will also be affected by compoundsolubility, polymer hydrophilicity, extent of polymer cross-linking,expansion of the polymer upon water absorption so as to make the polymerbarrier more permeable to the drug, geometry of the implant, and thelike. The implants are of dimensions commensurate with the size andshape of the region selected as the site of implantation. Implants maybe particles, sheets, patches, plaques, fibers, microcapsules and thelike and may be of any size or shape compatible with the selected siteof insertion.

The implants may be monolithic, i.e. having the active agenthomogenously distributed through the polymeric matrix, or encapsulated,where a reservoir of active agent is encapsulated by the polymericmatrix. The selection of the polymeric composition to be employed willvary with the site of administration, the desired period of treatment,patient tolerance, the nature of the disease to be treated and the like.Characteristics of the polymers will include biodegradability at thesite of implantation, compatibility with the agent of interest, ease ofencapsulation, a half-life in the physiological environment.

Biodegradable polymeric compositions which may be employed may beorganic esters or ethers, which when degraded result in physiologicallyacceptable degradation products, including the monomers Anhydrides,amides, orthoesters or the like, by themselves or in combination withother monomers, may find use. The polymers will be condensationpolymers. The polymers may be cross-linked or non-cross-linked. Ofparticular interest are polymers of hydroxyaliphatic carboxylic acids,either homo- or copolymers, and polysaccharides. Included among thepolyesters of interest are polymers of D-lactic acid, L-lactic acid,racemic lactic acid, glycolic acid, polycaprolactone, and combinationsthereof. By employing the L-lactate or D-lactate, a slowly biodegradingpolymer is achieved, while degradation is substantially enhanced withthe racemate. Copolymers of glycolic and lactic acid are of particularinterest, where the rate of biodegradation is controlled by the ratio ofglycolic to lactic acid. The most rapidly degraded copolymer has roughlyequal amounts of glycolic and lactic acid, where either homopolymer ismore resistant to degradation. The ratio of glycolic acid to lactic acidwill also affect the brittleness of in the implant, where a moreflexible implant is desirable for larger geometries. Among thepolysaccharides of interest are calcium alginate, and functionalizedcelluloses, particularly carboxymethylcellulose esters characterized bybeing water insoluble, a molecular weight of about 5 kD to 500 kD, etc.Biodegradable hydrogels may also be employed in the implants of thesubject invention. Hydrogels are typically a copolymer material,characterized by the ability to imbibe a liquid. Exemplary biodegradablehydrogels which may be employed are described in Heller in: Hydrogels inMedicine and Pharmacy, N. A. Peppes ed., Vol. III, CRC Press, BocaRaton, Fla., 1987, pp 137-149.

Pharmaceutical Dosages

Pharmaceutical compositions of the present disclosure containing ananti-C1q, anti-C1s, anti-C1r, and/or anti-C1 complex antibody of thepresent disclosure may be used (e.g., administered to an individual inneed of treatment with anti-C1q, anti-C1s, anti-C1r, and/or anti-C1complex antibody, preferably a human), in accordance with known methods,such as intravenous administration as a bolus or by continuous infusionover a period of time, by intramuscular, intraperitoneal,intracerobrospinal, intracranial, intraspinal, subcutaneous,intra-articular, intrasynovial, intrathecal, oral, topical, orinhalation routes.

Dosages and desired drug concentration of pharmaceutical compositions ofthe present disclosure may vary depending on the particular useenvisioned. The determination of the appropriate dosage or route ofadministration is well within the skill of an ordinary artisan. Animalexperiments provide reliable guidance for the determination of effectivedoses for human therapy. Interspecies scaling of effective doses can beperformed following the principles described in Mordenti, J. andChappell, W. “The Use of Interspecies Scaling in Toxicokinetics,” InToxicokinetics and New Drug Development, Yacobi et al., Eds, PergamonPress, New York 1989, pp. 42-46.

For in vivo administration of any of the anti-C1q, anti-C1s, anti-C1r,and/or anti-C1 complex antibodies of the present disclosure, normaldosage amounts may vary from about 10 ng/kg up to about 100 mg/kg of anindividual's body weight or more per day, preferably about 1 mg/kg/dayto 10 mg/kg/day, depending upon the route of administration. Forrepeated administrations over several days or longer, depending on theseverity of the disease, disorder, or condition to be treated, thetreatment is sustained until a desired suppression of symptoms isachieved.

An exemplary dosing regimen may include administering an initial dose ofan anti-C1q, anti-C1s, anti-C1r, and/or anti-C1 complex antibody, ofabout 2 mg/kg, followed by a weekly maintenance dose of about 1 mg/kgevery other week. Other dosage regimens may be useful, depending on thepattern of pharmacokinetic decay that the physician wishes to achieve.For example, dosing an individual from one to twenty-one times a week iscontemplated herein. In certain embodiments, dosing ranging from about 3μg/kg to about 2 mg/kg (such as about 3 μg/kg, about 10 μg/kg, about 30μg/kg, about 100 μg/kg, about 300 μg/kg, about 1 mg/kg, or about 2mg/kg) may be used. In certain embodiments, dosing frequency is threetimes per day, twice per day, once per day, once every other day, onceweekly, once every two weeks, once every four weeks, once every fiveweeks, once every six weeks, once every seven weeks, once every eightweeks, once every nine weeks, once every ten weeks, or once monthly,once every two months, once every three months, or longer. Progress ofthe therapy is easily monitored by conventional techniques and assays.The dosing regimen, including the anti-C1q, anti-C1s, anti-C1r, and/oranti-C1 complex antibody administered, can vary over time independentlyof the dose used.

Dosages for a particular anti-C1q, anti-C1s, anti-C1r, and/or anti-C1complex antibody may be determined empirically in individuals who havebeen given one or more administrations of the anti-C1q, anti-C1s,anti-C1r, and/or anti-C1 complex antibody. Individuals are givenincremental doses of an anti-C1q, anti-C1s, anti-C1r, and/or anti-C1complex antibody. To assess efficacy of an anti-C1q, anti-C1s, anti-C1r,and/or anti-C1 complex antibody, any clinical symptom of, for exampleGBS, can be monitored.

Administration of an anti-C1q, anti-C1s, anti-C1r, and/or anti-C1complex antibody of the present disclosure can be continuous orintermittent, depending, for example, on the recipient's physiologicalcondition, whether the purpose of the administration is therapeutic orprophylactic, and other factors known to skilled practitioners. Theadministration of an anti-C1q, anti-C1s, anti-C1r, and/or anti-C1complex antibody may be essentially continuous over a preselected periodof time or may be in a series of spaced doses.

Guidance regarding particular dosages and methods of delivery isprovided in the literature; see, for example, U.S. Pat. Nos. 4,657,760;5,206,344; or 5,225,212. It is within the scope of the invention thatdifferent formulations will be effective for different treatments anddifferent disorders, and that administration intended to treat aspecific organ or tissue may necessitate delivery in a manner differentfrom that to another organ or tissue. Moreover, dosages may beadministered by one or more separate administrations, or by continuousinfusion. For repeated administrations over several days or longer,depending on the condition, the treatment is sustained until a desiredsuppression of disease symptoms occurs. However, other dosage regimensmay be useful. The progress of this therapy is easily monitored byconventional techniques and assays.

Therapeutic Uses

The present disclosure provides anti-C1q, anti-C1s, anti-C1r, and/oranti-C1 complex antibodies which can bind to and neutralize a biologicactivity of C1q, C1s, C1r, and/or C1 complex. These anti-C1q, anti-C1s,anti-C1r, and/or anti-C1 complex antibodies are useful for preventing,reducing risk, or treating a wide range of autoimmune, such as GBS.Accordingly, as disclosed herein, anti-C1q, anti-C1s, anti-C1r, and/oranti-C1 complex antibodies of the present disclosure may be used fortreating, preventing, or reducing risk of GBS in an individual. In someembodiments, the individual has GBS. In some embodiments, the individualis a human.

As disclosed herein, “Guillain-Barré syndrome,” “GBS,” “Landry'sparalysis,” and “Guillain-Barr{acute over (w)}-Strohl syndrome” may beused interchangeably and refer to a disorder in which the body's immunesystem attacks part of the peripheral nervous system. The exact cause ofGuillain-Barre syndrome is unknown, but it is often preceded by aninfectious illness, such as a respiratory infection or the stomach flu.

The first symptoms of this disorder include varying degrees of weaknessor legs that tend to buckle, with or without tingling sensations in thelegs. In many instances the symmetrical weakness and abnormal sensationsspread, usually over periods of hours to days, to the arms, upper body,and facial muscles. Frequently, the lower cranial nerves may beaffected, leading to bulbar weakness, oropharyngeal dysphagia (drooling,or difficulty swallowing and/or maintaining an open airway) andrespiratory difficulties, and at times interference with blood pressureor heart rate. Most patients require hospitalization and about 30%require ventilatory assistance for treatment of type II respiratoryfailure. If present, sensory loss usually manifests as loss ofproprioception (position sense) and areflexia (complete loss of deeptendon reflexes), which is an important feature of GBS. Loss of pain andtemperature sensation is usually mild, as pain is a common symptom inGBS, presenting as deep aching pain, usually in the weakened muscles.These pains are self-limited and may be treated with standardanalgesics. Bladder dysfunction may also occur in severe cases.

Accordingly, the antibodies of the present disclosure may be used totreat, prevent, or improve one of more symptoms of GBS. In someembodiments, the present disclosure provides methods of treating,preventing, or improving one or more symptoms in individuals having GBS,by administering an anti-C1q, anti-C1s, anti-C1r, and/or anti-C1 complexantibody of the present disclosure to, for example, inhibit theinteraction between C1q and an autoantibody, such as an anti-gangliosideautoantibody, the interaction of C1q and C1r, and/or the interaction ofC1q and C1s. In some embodiments, the anti-C1q, anti-C1s, anti-C1r,and/or anti-C1 complex antibody inhibits C3c deposition. In someembodiments, the anti-C1q, anti-C1s, anti-C1r, and/or anti-C1 complexantibody inhibits membrane attack complex (MAC) deposition. In someembodiments, the anti-C1q, anti-C1s, anti-C1r, and/or anti-C1 complexantibody inhibits axonal damage formation. In some embodiments, theanti-C1q, anti-C1s, anti-C1r, and/or anti-C1 complex antibody inhibitsrespiratory muscle damage. The anti-C1q, anti-C1s, anti-C1r, and/oranti-C1 complex antibody may be administered to cells in vitro toprevent complement dependent cytotoxicity or complement-dependentcell-mediated cytotoxicity. The anti-C1q, anti-C1s, anti-C1r, and/oranti-C1 complex antibody may also be administered ex vivo in whole-mountmuscle models of GBS. Alternatively, the anti-C1q, anti-C1s, anti-C1r,and/or anti-C1 complex antibody may be administered in vivo (e.g., byadministering the antibody to an individual, such as a murine or humanindividual) to prevent to prevent C3c deposition or axonal damageformation.

Combination Treatments

The antibodies of the present disclosure may be used, withoutlimitation, in combination with any additional treatment for autoimmuneand/or neurodegenerative diseases, such as GBS, including, withoutlimitation, immunosuppressive therapies.

In some embodiments, an anti-C1q, anti-C1s, anti-C1r, and/or anti-C1complex antibody of this disclosure is administered in therapeuticallyeffective amounts in combination with a second neutralizinganti-complement factor antibody, such as an anti-C1q, anti-C1s,anti-C1r, and/or anti-C1 complex antibody, or a second anti-C1q,anti-C1s, anti-C1r, and/or anti-C1 complex antibody. In someembodiments, an anti-C1q, anti-C1s, anti-C1r, and/or anti-C1 complexantibody of this disclosure is administered in therapeutically effectiveamounts with a second and a third neutralizing anti-complement factorantibody, such as a second anti-C1q, anti-C1s, anti-C1r, and/or anti-C1complex antibody.

In some embodiments, the anti-C1q, anti-C1s, anti-C1r, and/or anti-C1complex antibodies of this disclosure are administered in combinationwith an inhibitor of antibody-dependent cellular cytotoxicity (ADCC).ADCC inhibitors may include, without limitation, soluble NK cellinhibitory receptors such as the killer cell Ig-like receptors (KIRs),which recognize HLA-A, HLA-B, or HLA-C and C-type lectin CD94/NKG2Aheterodimers, which recognize HLA-E (see, e.g., López-Botet M., T.Bellón, M. Llano, F. Navarro, P. Garcia & M. de Miguel. (2000), Pairedinhibitory and triggering NK cell receptors for HLA class I molecules.Hum. Immunol. 61: 7-17; Lanier L. L. (1998) Follow the leader: NK cellreceptors for classical and nonclassical MHC class I. Cell 92:705-707.), and cadmium (see, e.g., Immunopharmacology 1990; Volume 20,Pages 73-8).

In some embodiments, the antibodies of this disclosure are administeredin combination with an inhibitor of the alternative pathway ofcomplement activation. Such inhibitors may include, without limitation,factor B blocking antibodies, factor D blocking antibodies, soluble,membrane-bound, tagged or fusion-protein forms of CD59, DAF, CR1, CR2,Crry or Comstatin-like peptides that block the cleavage of C3,non-peptide C3aR antagonists such as SB 290157, Cobra venom factor ornon-specific complement inhibitors such as nafamostat mesilate (FUTHAN;FUT-175), aprotinin, K-76 monocarboxylic acid (MX-1) and heparin (see,e.g., T. E. Mollnes & M. Kirschfink, Molecular Immunology 43 (2006)107-121). In some embodiments, the antibodies of this disclosure areadministered in combination with an inhibitor of the interaction betweenthe autoantibody and its autoantigen. Such inhibitors may includepurified soluble forms of the autoantigen, or antigen mimetics such aspeptide or RNA-derived mimotopes, including mimotopes of the AQP4antigen. Alternatively, such inhibitors may include blocking agents thatrecognize the autoantigen and prevent binding of the autoantibodywithout triggering the classical complement pathway. Such blockingagents may include, e.g., autoantigen-binding RNA aptamers or antibodieslacking functional C1q binding sites in their Fc domains (e.g., Fabfragments or antibody otherwise engineered not to bind C1q).

Kits

The invention also provides kits containing antibodies of thisdisclosure for use in the methods of the present disclosure. Kits of theinvention may include one or more containers comprising a purifiedanti-C1q, anti-C1s, anti-C1r, and/or anti-C1 complex antibody andinstructions for use in accordance with methods known in the art.Generally, these instructions comprise a description of administrationof the inhibitor to treat or diagnose, e.g., GBS (such as an antibody ofthis disclosure), according to any methods known in the art. The kit mayfurther comprise a description of selecting an individual suitable fortreatment based on identifying whether that individual has the diseaseand the stage of the disease.

The instructions generally include information as to dosage, dosingschedule, and route of administration for the intended treatment. Thecontainers may be unit doses, bulk packages (e.g., multi-dose packages)or sub-unit doses. Instructions supplied in the kits of the inventionare typically written instructions on a label or package insert (e.g., apaper sheet included in the kit), but machine-readable instructions(e.g., instructions carried on a magnetic or optical storage disk) arealso acceptable.

The label or package insert indicates that the composition is used fortreating GBS. Instructions may be provided for practicing any of themethods described herein.

The kits of this invention are in suitable packaging. Suitable packagingincludes, but is not limited to, vials, bottles, jars, flexiblepackaging (e.g., sealed Mylar or plastic bags), and the like. Alsocontemplated are packages for use in combination with a specific device,such as an inhaler, nasal administration device (e.g., an atomizer) oran infusion device such as a minipump. A kit may have a sterile accessport (for example the container may be an intravenous solution bag or avial having a stopper pierceable by a hypodermic injection needle). Thecontainer may also have a sterile access port (e.g., the container maybe an intravenous solution bag or a vial having a stopper pierceable bya hypodermic injection needle). At least one active agent in thecomposition is an inhibitor of classical complement pathway. Thecontainer may further comprise a second pharmaceutically active agent.

Kits may optionally provide additional components such as buffers andinterpretive information. Normally, the kit comprises a container and alabel or package insert(s) on or associated with the container.

Diagnostic Uses

The antibodies of this disclosure also have diagnostic utility. Thisdisclosure therefore provides for methods of using the antibodies ofthis disclosure for diagnostic purposes, including the detection of C1q,C1s, C1r, and/or C1 complex in tissues, including tissues of a humanpatient. The anti-C1q, anti-C1s, anti-C1r, and/or anti-C1 complexantibodies of this disclosure are further useful for detecting synapsesand synapse loss, e.g., synapse loss experienced by patients sufferingfrom GBS. The phenomenon of synapse loss in neurodegeneration is wellunderstood in the art. See, e.g., U.S. Patent Publication Nos.2012/0195880 and 2012/0328601.

In some embodiments, the diagnostic methods involve the administrationof an anti-C1q, anti-C1s, anti-C1r, and/or anti-C1 complex antibody ofthis disclosure to an individual and the detection of antibody levelsbound to the synapses of the individual. Antibody binding to thesynapses of an individual can be quantitatively measured by non-invasivetechniques such as positron emission tomography (PET), X-ray computedtomography, single-photon emission computed tomography (SPECT), computedtomography (CT), and computed axial tomography (CAT).

In some embodiments, the diagnostic methods involve detecting synapsesin a biological sample, such as a biopsy specimen, a tissue, or a cell.An anti-C1q, anti-C1s, anti-C1r, and/or anti-C1 complex antibody iscontacted with the biological sample and the level of antibody bound tothe synapses present in the biological sample is then detected. Thedetection can be quantitative. Antibody detection in biological samplesmay occur with any method known in the art, including immunofluorescencemicroscopy, immunocytochemistry, immunohistochemistry, ELISA, FACSanalysis or immunoprecipitation.

Quantitation of synapse-bound antibodies provides a relativequantitative measure for the number of synapses present in theindividual. The diagnostic methods are typically repeated on a regularbasis, whereas the exact periodicity of the diagnostic measurementdepends on many factors, including the nature of the neurodegenerativedisease, the stage of disease progression, treatment modalities and manyother factors. Repeat diagnostic measurements typically revealprogressive synapse loss in patients suffering from neurodegenerativediseases. Synapse loss may be followed over time in individual patientssuffering from neurodegenerative diseases, but it may also be determinedin a diseased patient population relative to a healthy patientpopulation at any single point in time. Where patients are undergoing aspecific therapy, the relative loss of synapse numbers in individualsundergoing the specific therapy relative to the synapse loss observed inpatients not undergoing any treatments or undergoing control treatmentscan be used to assess the efficacy of the specific therapy provided.

The invention will be more fully understood by reference to thefollowing Examples. They should not, however, be construed as limitingthe scope of the invention. All citations throughout the disclosure arehereby expressly incorporated by reference.

EXAMPLES Example 1 Production of Anti-C1s Antibodies

The anti-C1s antibodies of this disclosure, including 5A1 and 5C12 (alsoreferred to as C1s^(mAb1) and C1s^(mAb2)), were generated by immunizingmice with human C1s enzyme purified from human plasma (ComplementTechnology Inc. Tyler Tex., Cat # A103) using standard mouseimmunization and hybridoma screening technologies (Milstein, C (1999).Bioessays 21: 966-73; Mark Page, Robin Thorpe, The Protein ProtocolsHandbook 2002, Editors: John M. Walker, pp 1111-1113). In brief, femaleBALB/c mice were injected intraperitoneal with 25 μg of protein incomplete Freund's adjuvant (CFA) on Day 0 and boosts were done with 25μg of C1s enzyme in incomplete Freund's adjuvant (IFA) on days 21, 42,52, and a final intravenous boost on day 63. Four days following thefinal boost the mice were euthanized, spleens removed, and splenocyteswere fused with the myeloma cell line SP2/0. Fused cells were grown onhypoxanthine-aminopterin-thymidine (HAT) selective semi-solid media for10-12 days and the resulting hybridomas clones were transferred to96-well tissue culture plates and grown in HAT medium until the antibodytitre is high. The antibody rich supernatants of the clones wereisolated and tested in an ELISA assay for reactivity with C1s. Positiveclones were isotyped and cultured for 32 days (post HAT selection) toidentify stable expressing clones.

Hybridoma cell lines producing anti-C1s antibodies 5A1 and 5C12 weredeposited with ATCC on May 15, 2013 having ATCC Accession NumbersPTA-120351 and PTA-120352. The anti-C1s antibodies 5A1 and 5C12 wereshown to bind to C1s and C1s-Pro and to neutralize biological functionsof C1s in cellular and biochemical assays (see, e.g., Examples 2-4).

Example 2 Anti-C1s Antibodies Specifically Bind to C1s and C1s-Pro

First, anti-C1s antibodies were screened for C1s and C1s proenzymebinding by ELISA.

ELISA assays were conducted using standard protocols. Briefly, theassays were conducted as follows. The day before the assay wasperformed, 96-well microtiter plates were coated at 0.2 μg/well ofC1s-enzyme antigen in 100 μL/well carbonate coating buffer pH9.6overnight at 4° C. Next, the plates were blocked with 3% milk powder inPBS for 1 hour at room temperature. Next, hybridoma tissue culturesupernatants were plated at 100 μL/well for 1 hour at 37° C. withshaking. The secondary antibody (1:10,000 goat anti-mouseIgG/IgM(H+L)-HRP) was applied at 100 μL/well for 1.5 hours at roomtemperature with shaking TMB substrate was added at 50 μL/well for 5minutes at room temperature in the dark. The reaction stopped with 50μL/well 1M HCl and read at 450 nm.

Six hybridoma supernatants (from 1B4, 3F8, 3G3, 5A1, 5C12, and 7C4) weretested for C1s and C1s proenzyme binding (FIG. 1). All six supernatantsshowed strong binding signals for the C1s proenzyme (middle column) aswell as the mature C1s protease (left column). Only background bindingsignals were observed with the negative control protein, humantransferrin (HT, right column) (FIG. 1).

These results showed that the antibodies produced by the hybridoma cells1B4, 3F8, 3G3, 5A1, 5C12, and 7C4 specifically bind C1s and the C1sproenzyme.

Example 3 Anti-C1s Antibodies Inhibit Complement-Mediated Hemolysis

Next, the ability of anti-C1s antibodies to neutralize cellularactivities of C1s was tested in a complement hemolytic assay.

A modified CH50 assay (also referred to as C1F hemolysis assay) wasperformed that provided limiting quantities of the C1 complex from humanserum to provide greater sensitivity for assessing C1 activity andpotential C1 inhibition. In brief, the assay was conducted as follows.First, 3×10⁷ sheep red blood cells (RBC) were incubated with anti-sheepRBC IgM antibody to generate activated erythrocytes (EA cells). The EAcells were then incubated with purified C4b protein to create EAC4bcells. EAC4b cells were subsequently incubated with diluted(1:1000-1:10000) normal human serum (NHS) that was pre-incubated with orwithout anti-C1s and control mouse IgG antibodies, to provide a limitingquantity of human C1. Next, the resulting EAC14 cells were incubatedwith purified human C2 protein to generate EAC14b2a cells. Finally,guinea pig serum was added in an EDTA buffer and incubated at 37° C. for30 minutes. Cell lysis was measured in a spectrophotometer at 450 nm.

Six C1s and C1s proenzyme binding antibodies (1B4, 3F8, 3G3, 5A1, 5C12,and 7C4) were tested for their ability to suppress complement mediatedhemolysis. Initial testing was conducted at a single antibodyconcentration (165 ng/ml). Out of the six antibodies tested, only 5A1and 5C12 antibodies showed substantial inhibition of hemolysis, whereas1B4, 3F8, 3G3, and 7C4 were essentially inactive (FIG. 2A). At antibodyconcentrations of 165 ng/ml, 5A1 suppressed 50% of the observedhemolysis and 5C12 suppressed 90% of hemolysis observed in the hemolyticassay (FIG. 2A). 5A1 and 5C12 were further tested in a dose-responseformat; both 5A1 and 5C12 were shown to inhibit hemolysis in adose-dependent manner (FIG. 2B).

These results demonstrate that not all C1s binding antibodies canneutralize complement mediated hemolysis. Two anti-C1s antibodies wereidentified, 5A1 and 5C12, that bind C1s and have substantial hemolysisneutralizing activity.

5A1 and 5C12 have also been shown to inhibit complement-dependent lysisof AQP4 expressing cells incubated with anti-AQP4 antibodies. Thisexperiment demonstrated the activity of the anti-C1s antibodies 5A1 and5C12 in a cellular model system of NMO and their utility for thetreatment of NMO. See U.S. Provisional Application No. 61/810,222 andPCT App. No. PCT/US2014/33560.

Example 4 Anti-C1s Antibodies Inhibit C1s-Mediated Cleavage of C4

To analyze the ability of anti-C1s antibodies to neutralize theproteolytic activity of C1s, 5A1 and 5C12 were tested for theirinhibitory activity on C1s-mediate cleavage of C4.

To this end, human C1s enzyme (2 ng; Complement Technology Inc., Catalog#A103) was incubated with an approximately 10-fold molar excess (25 ng)of C1s antibodies for 30 minutes at 4° C. Protein dilutions were made inPBS containing 0.1 mg gelatin/mL. The antibody/C1s mixture was incubatedwith 3 mg of human C4 protein (Complement Technology Inc. Cat # A105)for 5 minutes at 37° C. SDS-DTT Sample buffer was added to each sample,mixed and immediately placed in a 37° C. water bath for 15 min. Thesamples were loaded immediately onto a NuPage 10% BisTris SDS gel(Invitrogen Life Technologies) gel and ran for 1 hour at 150V. The gelwas fixed for 1 hour, stained with Coomassie Blue for 24 hrs andde-stained overnight.

Eight anti-C1s antibodies were tested in the C4 cleavage assay. 5A1 and5C12 inhibited C1s-mediated C4 α-chain cleavage if incubated atapproximately 10-fold molar excess, whereas six other anti-C1s bindingantibodies, including the C1s binding antibody 3F8 (see FIG. 1, Example2), did not show inhibitory activity (FIG. 3—upper panel). Furthertesting in a dose-response format demonstrated that both 5A1 and 5C12can inhibit C1s-mediated C4 cleavage at approximately equimolarconcentrations of antibody (3 ng) and C1s (2 ng; FIG. 3—lower panel).

Example 5 Characterization of Binding Epitope Mapping for Antibody M1Anti-C1q Antibody M1 Kinetic Analyses

Antibody M1 binding data was compared with corresponding data obtainedusing the reference antibody 4A4B11. The 4A4B11 antibody is described inU.S. Pat. No. 4,595,654. The 4A4B11 producing hybridoma cell line isavailable from ATCC (ATCC HB-8327TM).

C1q-antibody interactions were measured using an OCTET™ System accordingto standard protocols and manufacturer's instructions. Briefly, humanand mouse C1q proteins were immobilized separately on a biosensor atthree concentrations (3 nM, 1.0 nM, and 0.33 nM). Next, the anti-C1qantibody M1 was injected onto the C1q-coated biosensor at aconcentration of 2.0 μg/ml and the association constants (k_(on)) anddissociation constants (k_(off)) for anti-C1q antibodies M1 and 4A4B11were measured. The data were fit by non-linear regression analysis andusing the Octet Data Analysis software to yield affinity (K_(D)) andkinetic parameters (k_(on/off)) for the interactions of M1 and 4A4B11with human and mouse C1q respectively (Table 1).

TABLE 1 Kinetic Analysis of M1 and 4A4B11 Antibody Antigen K_(D) (M)k_(on) (1/Ms) k_(off) (1/s) M1 human C1q 1.28*10⁻¹¹ 5.18*10⁶ 6.31*10⁻⁵M1 mouse C1q 3.23*10⁻¹¹ 1.81*10⁶ 5.84*10⁻⁵ 4A4B11 human C1q 2.29*10⁻¹¹4.49*10⁶ 1.03*10⁻⁴ 4A4B11 mouse C1q undetectable undetectableundetectable

In this experimental series, anti-C1q antibody M1 was shown to bind bothhuman and mouse C1q proteins with very high affinities (K_(D)<10⁻¹⁰ M).By comparison, the reference antibody 4A4B11 was found to bind to humanC1q, whereas binding to mouse C1q was undetectable. Whereas theaffinities of M1 and 4A4B11 for human C1q were on the same order ofmagnitude (i.e., in the double-digit picomolar range; K_(D)˜10-30 pM),the affinity of M1 for mouse C1q was determined to be about four ordersof magnitude higher (K_(D)˜30 pM) than that the affinity of 4A4B11 formouse C1q (K_(D)˜40 nM).

Anti-C1q Antibodies M1 and 4A4B11 do not Compete for C1q-Binding

Blocking experiments were performed to determine whether the anti-C1qantibodies M1 and 4A4B11 bind to the same or overlapping epitopes ofhuman C1q or whether M1 and 4A4B11 bind to separate C1q epitopes.

To this end, M1 was coated on a biosensor chip (BIACORE™) andsubsequently contacted with a combination of human C1q and M1, acombination of human C1q and 4A4B11, or human C1q alone. C1q-binding toM1 was followed for 10 min and dissociation of M1-C1q complexes wassubsequently followed for 20 min. Relative binding signals were recordedat the end of the association and dissociation periods. Table 2 showsthe results of these experiments.

TABLE 2 Analysis of Simultaneous Interactions of M1 and 4A4B11 withhuman C1q Associa- Dissocia- Sensor Antigen Solution tion Response tionResponse Ab ID: ID: Ab ID: (nm) @600 s (nm) @1200 s M1 hC1q M1 −0.0119−0.00945 M1 hC1q 4A4B11 0.8213 0.82139 M1 hC1q None (Ag 0.4715 0.45137Only)

It was found that C1q alone bound effectively to immobilized M1 antibodyon the biosensor chip. Preincubation of C1q with soluble M1 antibodyprevented all binding of the resulting M1-C1q complex to immobilized M1.By contrast, preincubation of C1q with 4A4B11 did not prevent theinteraction of the resulting 4A4B11-C1q complex with immobilized M1. Thelarger relative binding signals observed in the binding experimentinvolving the 4A4B11-C1q complex relative to the binding experimentinvolving C1q alone is due to the fact that the relative binding signalscorrelate with the molecular weight of the soluble binding partners andthat the 4A4B11-C1q complex has a higher molecular weight than C1qalone.

These results demonstrate that 4A4B11 does not compete with M1 for C1qbinding. Therefore, 4A4B11 and M1 recognize separate epitopes on C1q.

Epitope Mapping

In order to determine the nature of the epitope (i.e. linear orconformational) bound by anti-C1q antibodies M1 and 4A4B11, theinhibition of the interaction between the C1Q protein and the antibodies4A4B11 (ANN-001) and M1 (ANN-005) by unstructured peptides generated byproteolysis of the C1q antigen was evaluated. If the peptides generatedby complete proteolysis of the antigen are able to inhibit the bindingof the antigen on the antibody, the interaction is not based onconformation, and the epitope is linear. If the peptides generated bycomplete proteolysis of the antigen are unable to inhibit the binding ofthe antigen on the antibodies 4A4B11 and M1, the conformation isnecessary for interaction. Based on the data described in detail below,unstructured peptides generated by digestion of native C1q did notcompete with intact C1q for binding to the 4A4B11 (ANN-001) and M1(ANN-005) antibodies (FIG. 4), suggesting that the C1q epitope for theseantibodies is a complex conformational epitope.

In order to determine the key residues of the conformational C1q epitopethat binds of ANN-001 and ANN-005 on C1Q antigen with high resolutionthe antibody/antigen complexes were incubated with deuteratedcross-linkers and subjected to multi-enzymatic proteolytic cleavage.After enrichment of the cross-linked peptides, the samples were analyzedby high resolution mass spectrometry (nLC-Orbitrap MS) and the datagenerated analyzed using XQuest software. The analysis described belowindicates that antibody 4A4B11 (ANN-001) binds to an epitope thatincludes amino acids 5202 and K219 of human C1QA and Y225 of human C1QC,and antibody M1 (ANN-005) binds to an epitope that includes amino acidK219 of human C1QA and S185 of human C1QC. See the amino acid sequencealignment of human and mouse C1qA and C1qC as shown below.

Amino acid sequence alignment of human and mouse C1qAMEGPRGWLVLCVLAISLASMVTEDLCRAPDGKKGEAGRPGRRGRPGLKGEQGEPGAPGIR humanMETSQGWLVACVLTMTLVWTVAEDVCRAPNGKDGAPGNPGRPGRPGLKGERGEPGAAGIR mouse** .:**** ***:::*.  *:**:****:**.* .*.*** ********:*****.***TGIQGLKGDQGEPGPSGNPGKVGYPGPSGPLGARGIPGIKGTKGSPGNIKDQPRPAFSAI humanTGIRGFKGDPGESGPPGKPGNVGLPGPSGPLGDSGPQGLKGVKGNPGNIRDQPRPAFSAI mouse***:*:*** **.**.*:**:** ********  *  *:**.**.****:**********RRNPPMGGNVVIFDTVITNQEEPYQNHSGRFVCTVPGYYYFTFQVLSQWEICLSIVSSSR humanRQNPMTLGNVVIFDKVLTNQESPYQNHTGRFICAVPGFYYFNFQVISKWDLCLFIKSSSG mouse*:** *********.*:****.*****:***:*:***:***.***:*:*::** * ***GQVRRSLGFCDTTNKGLFQVVSGGMVLQLQQGDQVWVEKDPKKGHIYQGSEADSVFSGFL humanGQPRDSLSFSNTNNKGLFQVLAGGTVLQLRRGDEVWIEKDPAKGRIYQGTEADSIFSGFL mouse** * **.*.:*.*******::** ****::**:**:**** **:****:****:*****IFPSA humanSEQ ID NO: 1 IFPSA mouseSEQ ID NO: 6 *****Amino acid sequence alignment of human and mouse C1qCMDVGPSSLPHLGLKLLLLLLLLP-LRGQANTGCYGIPGMPGLPGAPGKDGYDGLPGPKGE humanMVVGPSCQPPCGLCLLLLFLLALPLRSQASAGCYGIPGMPGMPGAPGKDGHDGLQGPKGE mouse* ****. ** ** ****:**   **.**.:**********:********:*** *****PGIPAIPGIRGPKGQKGEPGLPGHPGKNGPMGPPGMPGVPGPMGIPGEPGEEGRYKQKFQ humanPGIPAVPGTRGPKGQKGEPGMPGHRGKNGPRGTSGLPGDPGPRGPPGEPGVEGRYKQKHQ mouse*****:** ***********:*** ***** *  *:** *** * ***** ******* *SVFTVTRQTHQPPAPNSLIRFNAVLTNPQGDYDTSTGKFTCKVPGLYYFVYHASHTANLC humanSVFTVTRQTTQYPEANALVRFNSVVTNPQGHYNPSTGKFTCEVPGLYYFVYYTSHTANLC mouse********* * * .*:*:***:*:*****.*:.*******:*********::*******VLLYRSGVKVVTFCGHTSKTNQVNSGGVLLRLQVGEEVWLAVNDYYDMVGIQGSDSVFSG humanVHLNLNLARVASFCDHMFNSKQVSSGGVLLRLQRGDEVWLSVNDYNGMVGIEGSNSVFSG mouse* *  . .:*.:**.*  :::**.********* *;****;**** .****:**:*****FLLFPD human SEQ ID NO: 3 FLLFPD mouse SEQ ID NO: 7 ******

1. Identification of the C1q/Antibody Complexes by Mass Spectrometry

The C1q/antibody complexes were generated by mixing equimolar solutionsof C1q antigen and antibody (4 μM in 5 μl each). One μl of the mixtureobtained was mixed with 1 μl of a matrix composed of a re-crystallizedsinapinic acid matrix (10 mg/ml) in acetonitrile/water (1:1, v/v), TFA0.1% (K200 MALDI Kit). After mixing, 1 μl of each sample was spotted onthe MALDI plate (SCOUT 384). After crystallization at room temperature,the plate was introduced in the MALDI mass spectrometer and analysedimmediately. The analysis has been repeated in triplicate. FIG. 4 showsthe presence of the antigen, antibody and antigen/antibody complexes forC1q/4A4B11 (FIG. 4A) and C1q/M1 (FIG. 4B). Peaks are present at thepredicted molecular weights of monomeric antibody (˜150 kDa) and C1qmonomer (˜460 kDa) and there is a 1:1 complex of antibody:antigenpresent at ˜615 kDa.

2. Unstructured C1q Peptides Generated by Proteolysis do not Compete forBinding of C1q to Antibody

To determine if the C1q/antibody complexes could be competed withpeptides the C1q antigen was digested with immobilized pepsin. 25 μl ofthe antigen with a concentration of 10 μM were mixed with immobilizedpepsin 5 μM and incubate at room temperature for 30 minutes. After theincubation time the sample was centrifuged and the supernatant waspipetted. The completion of the proteolysis was controlled by High-MassMALDI mass spectrometry in linear mode. The pepsin proteolysis wasoptimized in order to obtain a large amount of peptide in the 1000-3500Da range. Next, 5 μl of the antigen peptides generated by proteolysiswere mixed with 5 μl of ANN-001 or ANN-005 (8 μM) and incubated at 37°for 6 hours. After incubation of ANN-001 or ANN-005 with the C1Q antigenpeptides, 5 μl of the mixture was mixed with 5 μl of the C1Q antigen (4μM) so the final mix contained 2 μM/2 μM/2.5 μM of C1Q antigen/4A4B11 orM1/C1Q antigen Peptides.

The MALDI ToF MS analysis was performed using CovalX's HM3 interactionmodule with a standard nitrogen laser and focusing on different massranges from 0 to 2000 kDa. For the analysis, the following parametershave been applied for Mass Spectrometer: Linear and Positive mode; IonSource 1: 20 kV; Ion Source 2: 17 kV; Pulse Ion Extraction: 400 ns; forHM3: Gain Voltage: 3.14 kV; Gain Voltage: 3.14 kV; Acceleration Voltage:20 kV.

To calibrate the instrument, an external calibration with clusters ofInsulin, BSA and IgG has been applied. For each sample, 3 spots wereanalyzed (300 laser shots per spots). The presented spectrum correspondsto the sum of 300 laser shots. The MS data were analyzed using theComplex Tracker analysis software version 2.0 (CovalX Inc).

3. Identification of the Conformational Epitopes for C1q Binding toANN-001 and ANN-005

Using chemical cross-linking, High-Mass MALDI mass spectrometry andnLC-Orbitrap mass spectrometry the interaction interface between theantigen C1Q and two monoclonal antibodies ANN-001 and ANN-005 wascharacterized. 5 μl of the sample C1Q antigen (concentration 4 μM) wasmixed with 5 μl of the sample ANN-001 (Concentration 4 μM) or ANN-005(Concentration 4 μM) in order to obtain an antibody/antigen mix withfinal concentration 2 μM/2 μM. The mixture was incubated at 37° C. for180 minutes. In a first step, 1 mg of DiSuccinimidylSuberate H12(DSS-H12) cross-linker was mixed with 1 mg of DiSuccinimidylSuberate D12(DSS-D12) cross-linker. The 2 mg prepared were mixed with 1 ml of DMF inorder to obtain a 2 mg/ml solution of DSS H12/D12. 10 μl of theantibody/antigen mix prepared previously were mixed with 1 μl of thesolution of cross-linker d0/d12 prepared (2 mg/ml). The solution wasincubated 180 minutes at room temperature in order to achieve thecross-linking reaction. In order to facilitate the proteolysis, it wasnecessary to reduce the disulfide bound present in this protein. Thecross-linked sample was mixed with 20 μl of ammonium bicarbonate (25 mM,pH 8.3). After mixing 2.5 μl of DTT (500 mM) is added to the solution.The mixture was then incubated 1 hour at 55° C. After incubation, 2.5 μlof iodoacetamide (1M) was added before 1 hour of incubation at roomtemperature in a dark room. After incubation, the solution was diluted1/5 by adding 120 μl of the buffer used for the proteolysis. 145 μl ofthe reduced/alkyled cross-linked sample was mixed with 2 μl of trypsin(Sigma, T6567). The proteolytic mixture was incubated overnight at 37°C. For α-chymotrypsin proteolysis, the buffer of proteolysis wasTris-HCL 100 mM, CaCl₂10 mM, pH7.8. The 145 μl of the reduced/alkyledcross-linked complex was mixed with 2 μl of α-chymotrypsin 200 μM andincubated overnight at 30° C. For this analysis, an nLC in combinationwith Orbitrap mass spectrometry were used. The cross-linker peptideswere analyzed using Xquest version 2.0 and stavrox software. Thepeptides identified and cross-linked amino acids are indicated in Table3 below.

TABLE 3C1q cross-linked peptides and contact residues necessary for ANN-001 and ANN-005 binding Protease Contact Digest X-linked Peptide C1q SubunitResidue Antibody Trypsin GLFQVVSGGMVLQLQQGDQVWVE C1qA K219 ANN-001K(SEQ ID NO: 8) Trypsin FQVVSGGMVLQL (SEQ ID NO: 9) C1qA 5202 ANN-001Chymotrypsin YDMVGIQGSDSVFSGF C1qC Y225 ANN-001 (SEQ ID NO: 10 TrypsinGLFQVVSGGMVLQLQQGDQVWVE C1qA K219 ANN-005 K (SEQ ID NO: 11) ChymotrypsinRSGVKVVTF (SEQ ID NO: 12) C1qC 5185 ANN-005

Example 6 Materials and Methods Antibodies and ImmunohistologicalAnalysis

The IgM anti-GQ1b ganglioside mAb, CGM3 was derived from mice inoculatedwith a GT1a-bearing C. jejuni lipooligosaccharide (Goodyear et al.,1999). CGM3 reacts with gangliosides GQ1b, GD3, and GT1a that all sharethe terminal disialylgalactose structure. Previous in vitro studies haveshown that CGM3 has similar ganglioside specificity and inducesidentical complement dependent pathogenic effects as human MFS sera(Goodyear et al., 1999; Plomp et al., 1999; Bullens et al., 2000; Jacobset al., 2002; Jacobs et al., 2003). The control IgG1 antibody is a mousemonoclonal antibody obtained from Antibody Solutions (Cat #AP1-C).Normal human serum (NHS) was taken from a single donor stock that hadbeen freshly frozen and stored in multiple aliquots at −70° C. topreserve complement activity. Prior to experimental use, CGM3 and NHSwere dialysed for 24 h at 4° C. against Ringer's solution (116 mM NaCl,4.5 mM KCl, 1 mM MgCl₂, 2 mM CaCl₂, 1 mM NaH₂PO₄, 23 mM NaHCO₃, 11 mMglucose, pH 7.4) and pre-gassed with 95% O₂/5% CO₂.

The intermediate complement component C3c was detected by incubationwith FITC-labelled rabbit anti-C3c (1/300; Dako, Ely, UK); Forneurofilament staining, sections of unfixed tissue were preincubated for1 h at 4° C. with TRITC-conjugated a-BTx to label the nAChR at the NMJ,rinsed, immersed in ethanol at −20° C. for 20 min, then incubatedovernight at room temperature with the rabbit polyclonal serum 1211(1/750; reactive with phosphorylated neurofilament; Affiniti ResearchProducts Ltd. Exeter, UK) followed by FITC conjugated goat anti-rabbitIgG (1/300; Southern Biotechnology Associates) for 3 h at 4° C. Alldetection antibodies were diluted in phosphate buffered saline (PBS).

Ex Vivo Whole-Mount Muscle-Nerve Model of GBS

Mouse hemi-diaphragms with phrenic nerve attached (or triangularissterni muscle in some cases for illustrative NMJ immunohistology) weredissected and mounted in Ringer's medium at room temperature (20-22°C.). Untreated small control sections were removed from each musclepreparation prior to any incubations and snap frozen on dry ice forsubsequent baseline immunohistological analysis. Muscles were incubatedwith the anti-ganglioside monoclonal antibody CGM3 (50 μg/ml) for 2-2.5h at 32° C., then for 30 min at 4° C. and then equilibrated for 10 minat room temperature, rinsed in Ringer's medium and subsequently exposedto 40% NHS in Ringer's medium for 1 h at room temperature. C1 antibodies(100 μg/ml) or the control mAb (100 μg/ml) was mixed with NHS 10 minprior to the incubation of the muscle preparation. The intermediatecomplement component C3c was detected by incubation with FITC-labelledrabbit anti-C3c (1/300; Dako, Ely, UK) for 1 h at 4° C. Digital imageswere captured using both a Zeiss Pascal confocal laser scanningmicroscope and a Zeiss Axio Imager Z1 with ApoTome. Image-analysismeasurements were made using ImageJ (NIH, USA) image analysis software.For quantitative analysis of C3c, MAC and Nfil three staining runs ofeach marker were performed on tissue from at least three individualhemi-diaphragms, and quantified as previously described (O'Hanlon etal., 2001).

In Vivo Model of GBS

Balb/c mice (3-4 weeks old, 10-15 g) were injected intraperitoneallywith 1.5 mg CGM3, followed 16 h later by an intravascular (IV) injectionof 200 mg of anti-C1q (i.e., M1) antibody and intraperitoneal (IP)injection of 0.5 ml 100% NHS. After 4 hours the mice were euthanizedwith CO₂ and diaphragm muscle tissue was dissected and processed forimmunohistological analyses (see Examples 7-9 below). The in vivo effectof the anti-C1q antibodies 4A4B11 and M1 on diaphragm C3c deposition andaxonal integrity was determined.

Production and Characterization of Anti-C1q and Anti-C1s Antibodies

The anti-C1q antibody 4A4B11 is described in U.S. Pat. No. 4,595,654 andthe hybridoma for this line is available from ATCC (ATCC HB-8327TM). Theanti-C1q antibody M1 was generated by Antibody Solutions Inc. (SunnyvaleCalif.) by immunizing C1q knockout mice with human C1q using standardmouse immunization and hybridoma screening technologies (Milstein, C(1999). Bioessays 21: 966-73; Mark Page, Robin Thorpe, The ProteinProtocols Handbook 2002, Editors: John M. Walker, pp 1111-1113). Thegeneration and characterization of this antibody is described in USpatent application (Ser. No. 61/844,368).

The anti-C1s antibody 5C12 was generated and characterized as describedin Example 1 above.

Example 7 C1 Antibodies Prevent C3c Deposition in an Ex Vivo Whole-MountMuscle Model of GBS

Anti-C1q and anti-C1s antibody efficacy was tested in an ex vivodiaphragm model of GBS in which muscles with phrenic nerve attached wereincubated with the anti-ganglioside monoclonal antibody CGM3 (50 mg/ml)for 2-2.5 h at 32° C., then for 30 min at 4° C., and then equilibratedfor 10 min at room temperature, rinsed in Ringer's medium, andsubsequently exposed to 40% NHS in Ringer's medium for 1 h at roomtemperature. C1 antibodies (100 μg/ml) or the control mAb (100 μg/ml)was mixed with NHS 10 min prior to the incubation of the musclepreparation. An anti-ganglioside monoclonal antibody and humancomplement (i.e., Ab+NHS) causes complement deposition and damage ofmotor neuron axons. FIG. 6 shows that incubation of the anti-C1q mAbwith the Ab+NHS causes a statistically significant reduction in C3cdeposition relative to all of the other groups. The 5C12 antibody showsa reduction in C3c deposition but it does not appear to be statisticallysignificant in this experiment.

It was also demonstrated that anti-C1q antibodies 4A4B11 and M1 cansuppress complement deposition and preserve axonal integrity in theex-vivo diaphragm model of GBS. FIG. 7A shows a box and whisker plot ofthe quantitation of the immunofluorescent labeling of C3c deposition onthe explanted diaphragm. The control antibody showed a statisticallysignificant increase in C3c staining compared to the untreated tissue(*−p<0.01; Mann-Whitney test), while the anti-C1q antibodies were notdifferent than untreated tissue (FIG. 7A). FIG. 7B shows images ofsections quantitated in FIG. 7A. The tissues were either untreated (nocomplement added) or treated with a control IgG1 antibody or theanti-C1q antibodies 4A4B11 and M1. The results demonstrate that additionof anti-C1q antibodies resulted in almost complete elimination of C3cdeposition at the NMJ (FIG. 7B), compared to tissue treated with thecontrol antibody (FIG. 7B, left column). FIG. 7C shows quantitation ofthe neurofilament staining at the NMJ, demonstrating that anti-C1qantibody treatment causes a statistically significant increase, comparedto the control antibody, in neurofilament staining representative ofincreased axonal integrity. FIG. 7D shows representative images of NMJshowing the post-synaptic membrane (nAChR) and axon (Nfil) staining inthe presence of the control antibody and the anti-C1q antibodies. Theimages demonstrate that the diminished axonal neurofilament labeling insamples treated with the control antibody treated samples issignificantly restored by treatment with the anti-C1q antibodies (FIG.7D). Both the anti-C1q antibody M1 and the anti-C1q antibody 4A4B11caused a statistically significant increase in neurofilament staining(i.e., axonal integrity) relative to the control IgG1 antibody(*/#—p<0.01; Mann-Whitney test) (FIG. 7C).

Example 8 C1q Antibody M1 Prevents Axonal Damage Formation in an In VivoMouse Model of GBS

The efficacy of the anti-C1q antibody M1 was tested in an in vivo mousemodel of GBS in which 1.5 mg anti-ganglioside antibody (n=7) wasinjected by IP and 16 h later, IV injection of 200 mg M1 (˜15 mg/kg)(n=4) or IgG1 control antibody (Antibody Solutions AP1-C) (n=3) followedby IP administration of 100% normal human serum. After 4 hours diaphragmtissue was collected and processed for C3c deposition, and axonalintegrity over the endplate was assessed by neurofilament staining witha polyclonal rabbit antibody to phosphorylated neurofilament. MannWhitney test was used to assess the statistical significance of theresults (***=p<0.001). FIG. 8A shows a box and whisker plot of thequantitation of the C3c immunofluorescence at the motor nerve endplateand below the graph is an image of C3c (green) deposition at the NMJwith muscle fluorescently labeled by α-bungarotoxin (BTx; red). Thisimage demonstrates the inhibition of C3c deposition by the M1 antibody.FIG. 8B shows a box and whisker plot of the quantitation of the MACimmunofluorescence at the motor nerve endplate and a corresponding imageof MAC (green) deposition at the NMJ with muscle fluorescently labeledby BTx (red) demonstrating that M1 treatment causes blockade of MACdeposition and thus blockade of complement factors upstream (C3c) anddownstream (MAC) of the C5 protein, which is the target of Eculizumabfunction. FIG. 8C shows quantitation of the neurofilament staining atthe NMJ and an image of the neorofilament staining demonstrating that M1treatment causes a dramatic increase in neurofilament stainingrepresentative of increased axonal integrity.

Example 9 C1q Antibody M1 Decreases Respiratory Muscle Damage in an InVivo Mouse Model of GBS

The efficacy of the anti-C1q antibody M1 in treating respiratory muscledamage was tested in an in vivo mouse model of GBS.

In this in vivo mouse model of GBS, it is a part of the autoimmunedisease process that attacks the motor nerve terminals at the diaphragmand blocks normal nerve transmission to the diaphragm. Injury wasinduced in seven mice by injecting the mice by intraperitoneal (IP)injection with 1.5 mg anti-ganglioside antibody combined with serum. Thefollowing day mice were injected by IV injection with 50 mg/kg ofanti-C1q antibody M1 (n=4) or isotype control antibody ACP1 (AntibodySolutions, Mountain View, Calif.) (n=3), followed by IP injection ofnormal human serum (NHS) 30 min later. Baseline tidal volume readingswere recorded the day before antibody injection and subsequent readingswere taken at 4 h and 6 h post-injury induction (FIG. 9A).

FIG. 9B shows the percentage change from baseline tidal volume for theexperimental groups. The results depicted in FIG. 9 demonstrate thattidal volume is significantly reduced, compared to baseline volume, inmice treated with the isotype control ACP-1 antibody. However, normaltidal volumes were maintained in mice treated with the M1 antibody.

Example 10 Test for C1q Antibody as a Therapeutic Agent in a GBSClinical Trial

The efficacy of a humanized C1q antibody is tested in a clinical trialfor GBS as previously described (e.g., Clinical Trials website for GBSstudy with Eculizumab).

Briefly, the incidence of adverse effects and severe adverse is firstdetermined with the humanized anti-C1q antibody and IVIg compared toplacebo controls over a 6-18 month period following intravenousdelivery. Improvement of one or more grade in functional outcome (on the6 point GBS disability scale) is then monitor at 4 weeks. The ability towalk unaided (GBS disability score 2) is also monitored at 8 weeks. Thefollowing effects are also monitored: time taken to improve by at leastone grade (on the GBS disability scale) at 8 weeks, the time taken towalk independently at 1 year, difference in GBS disability score atmaximum disability completed with 6 months, percentage of patients witha clinically relevant improvement in R-ODS score at 6 months, increasefrom baseline in R-ODS score by at least 6 points on the centile metricscore at 4 weeks and 6 months, percentage of patients with a clinicallyrelevant improvement in ONLS at 6 months (defined as an increase frombaseline in ONLS score by at least 1 point at 4 weeks and 6 months),requirement for ventilatory support (GBS disability score 5) over 4weeks, duration of ventilatory support over 8 weeks, occurrence ofrelapse over 2 years, and mortality within the first 6 months.

Eligibility criteria include, without limitation: age of 18 years andolder; both genders; patients aged >18 years diagnosed with GBSaccording to NINDS diagnostic criteria; onset of weakness due to GBS isless than 2 weeks ago; patients who are unable to walk unaided for >10meters (grade >3 on GBS disability scale); patients who are beingconsidered for or already on IVIg treatment. The first dose of anti-C1qantibody treatment may be started within 2 weeks from onset of weaknessand any time during the IVIg treatment period. Signed informed consentis also obtained.

Exclusion criteria include, without limitation: age <18 years; patientswho are pregnant or lactating; patients that show clear clinicalevidence of a polyneuropathy caused by e.g., diabetes mellitus (exceptmild sensory), alcoholism, severe vitamin deficiency, and porphyria;patients having received immunosuppressive treatment (e.g.,azathioprine, cyclosporine, mycofenolatemofetil, tacrolimus, sirolimusor >20 mg prednisolone daily) during the previous month; patients knownto have severe concurrent disease, like malignancy, severecardiovascular disease, AIDS, severe COPD, TB, etc.; inability to complywith study related procedures or appointments during 6 months; anycondition that in the opinion of the investigator could increase thepatient's risk by participating in the study or confound the outcome ofthe study; unresoled Neisseria meningitidis infection or history ofmeningococcal infection; patients unsuitable for antibiotic prophylaxis(e.g., due to allergy); known hypersensitivity to anti-C1q antibody,murine proteins, or to any of the excipients; patients known orsuspected of hereditary complement deficiencies, and women ofchild-bearing potential who are unwilling to use effective contraceptionduring treatment and for 5 months after treatment is completed.

Deposit of Material

The following materials have been deposited according to the BudapestTreaty in the American Type Culture Collection, ATCC Patent Depository,10801 University Blvd., Manassas, Va. 20110-2209, USA (ATCC):

Deposit ATCC Sample ID Isotype Date Accession No. Mouse anti-C1s-RP mABcell line IgG1, May 15, PTA-120351 5A1 IgG1 producing antibody 5A1 kappa2013 Mouse anti-C1s-RP mAb cell line IgG1, May 15, PTA-120352 5C12 IgG1producing antibody 5C12 kappa 2013 Mouse hybridoma C1qM1 7788-1(M) IgG1,Jun. 6, PTA-120399 051613 producing anti-C1q antibody kappa 2013 M1

The hybridoma cell lines producing the 5A1 antibody (mouse anti-C1s-RPmAb cell line 5A1 IgG1), the 5C12 antibody (mouse anti-C1s-RP mAb cellline 5C12 IgG1), and M1 antibody (mouse hybridoma C1qM1 7788-1(M)051613) have each been deposited with ATCC under conditions that assurethat access to the culture will be available during pendency of thepatent application and for a period of 30 years, or 5 years after themost recent request, or for the effective life of the patent, whicheveris longer. A deposit will be replaced if the deposit becomes nonviableduring that period. Each of the deposits is available as required byforeign patent laws in countries wherein counterparts of the subjectapplication, or its progeny are filed. However, it should be understoodthat the availability of the deposits does not constitute a license topractice the subject invention in derogation of patent rights granted bygovernmental action.

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1. A method of treating or preventing Guillain-Barré Syndrome (GBS),comprising administering to an individual an antibody, wherein theantibody is: a) an anti-C1q antibody, wherein the anti-C1q antibodyinhibits the interaction between C1q and an autoantibody, or between C1qand C1r, or between C1q and C1s, or wherein the anti-C1q antibodyprevents C1q from activating C1r or C1s; b) an anti-C1s antibody,wherein the anti-C1s antibody inhibits the interaction between C1s andC1q, or between C1s and C1r, or between C1s and C2, or between C1s andC4, or wherein the anti-C1s antibody inhibits the catalytic activity ofC1s or inhibits the processing of pro-C1s to an active protease; c) ananti-C1r antibody, wherein the anti-C1r antibody inhibits theinteraction between C1r and C1q, or between C1r and C1s, or wherein theanti-C1r antibody inhibits the catalytic activity of C1r or inhibits theprocessing of pro-C1r to an active protease; or d) an anti-C1 complexantibody that binds to a combinatorial epitope within the C1 complex,wherein said combinatorial epitope is comprised of C1q and C1s; C1q andC1r; C1r and C1s; or C1q, C1r, and C1s; or wherein the anti-C1 complexantibody inhibits C1r or C1s activation or prevents their ability to acton C2 or C4.
 2. The method of claim 1, wherein the antibody binds C1q,C1r, or C1s.
 3. The method of claim 1, wherein the antibody is ananti-C1q antibody.
 4. The method of claim 3, wherein the anti-C1qantibody is: a) an anti-C1q antibody comprising a light chain variabledomain and a heavy chain variable domain, wherein the light chainvariable domain comprises the HVR-L1, HVR-L2, and HVR-L3 of themonoclonal antibody M1 produced by a hybridoma cell line with ATCCAccession Number PTA-120399 or progeny thereof; and/or wherein the heavychain variable domain comprises the HVR-H1, HVR-H2, and HVR-H3 of themonoclonal antibody M1 produced by a hybridoma cell line with ATCCAccession Number PTA-120399 or progeny thereof; or b) an anti-C1qantibody which binds essentially the same C1q epitope as the antibody M1produced by the hybridoma cell line with ATCC Accession NumberPTA-120399 or anti-C1q binding fragments thereof.
 5. The method of claim3, wherein the anti-C1q antibody is an anti-C1q antibody that binds to aC1q protein and binds to one or more amino acids of the C1q proteinwithin amino acid residues selected from: a) amino acid residues 196-226of SEQ ID NO:1, or amino acid residues of a C1 q protein chain A (C1qA)corresponding to amino acid residues 196-226(GLFQVVSGGMVLQLQQGDQVWVEKDPKKGHI) of SEQ ID NO:1; b) amino acid residues196-221 of SEQ ID NO:1, or amino acid residues of a C1qA correspondingto amino acid residues 196-221 (GLFQVVSGGMVLQLQQGDQVWVEKDP) of SEQ ID.NO:1; c) amino acid residues 202-221 of SEQ ID NO:1, or amino acidresidues of a C1qA corresponding to amino acid residues 202-221(SGGMVLQLQQGDQVWVEKDP) of SEQ ID NO:1; d) amino acid residues 202-219 ofSEQ ID NO:1, or amino acid residues of a C1qA corresponding to aminoacid residues 202-219 (SGGMVLQLQQGDQVWVEK) of SEQ ID NO:1; e) amino acidresidues Lys 219 and/or Ser 202 of SEQ ID NO:1, or amino acid residuesof a C1qA corresponding Lys 219 and/or Ser 202 of SEQ ID NO:1; f) aminoacid residues 218-240 of SEQ ID NO:3 or amino acid residues of a C1qprotein chain C (C1qC) corresponding to amino acid residues 218-240(WLAVNDYYDMVGIQGSDSVFSGF) of SEQ ID NO:3; g) amino acid residues 225-240of SEQ ID NO:3 or amino acid residues of a C1qC corresponding to aminoacid residues 225-240 (YDMVGI QGSDSVFSGF) of SEQ ID NO:3; h) amino acidresidues 225-232 of SEQ ID NO:3 or amino acid residues of a C1qCcorresponding to amino acid residues 225-232 (YDMVGIQG) of SEQ ID NO:3;i) amino acid residue Tyr 225 of SEQ ID NO:3 or an amino acid residue ofa C1qC corresponding to amino acid residue Tyr 225 of SEQ ID NO:3; j)amino acid residues 174-196 of SEQ ID NO:3 or amino acid residues of aC1qC corresponding to amino acid residues 174-196(HTANLCVLLYRSGVKVVTFCGHT) of SEQ ID NO:3; k) amino acid residues 184-192of SEQ ID NO:3 or amino acid residues of a C1qC corresponding to aminoacid residues 184-192 (RSGVKVVTF) of SEQ ID NO:3; l) amino acid residues185-187 of SEQ ID NO:3 or amino acid residues of a C1qC corresponding toamino acid residues 185-187 (SGV) of SEQ ID NO:3; and m) amino acidresidue Ser 185 of SEQ ID NO:3 or an amino acid residue of a C1qCcorresponding to amino acid residue Ser 185 of SEQ ID NO:3. 6.(canceled)
 7. The method of claim 3, wherein the anti-C1q antibody is ananti-C1q antibody that binds to a C1q protein and binds to one or moreamino acids of the C1q protein chain A (C1qA) within amino acid residuesselected from: a) amino acid residues 196-226 of SEQ ID NO:1, or aminoacid residues of a C1q protein chain A (C1qA) corresponding to aminoacid residues 196-226 (GLFQVVSGGMVLQLQQGDQVWVEKDPKKGHI) of SEQ ID NO:1;b) amino acid residues 196-221 of SEQ ID NO:1, or amino acid residues ofa C1qA corresponding to amino acid residues 196-221(GLFQVVSGGMVLQLQQGDQVWVEKDP) of SEQ ID. NO:1; c) amino acid residues202-221 of SEQ ID NO:1, or amino acid residues of a C1qA correspondingto amino acid residues 202-221 (SGGMVLQLQQGDQVWVEKDP) of SEQ ID NO:1; d)amino acid residues 202-219 of SEQ ID NO:1, or amino acid residues of aC1qA corresponding to amino acid residues 202-219 (SGGMVLQLQQGDQVWVEK)of SEQ ID NO:1; and e) amino acid residue Lys 219 of SEQ ID NO:1, or anamino acid residue of a C1qA corresponding Lys 219 of SEQ ID NO:1; andwherein the anti-C1q antibody binds to one or more amino acids of theC1q protein chain C (C1qC) within amino acid residues selected from: f)amino acid residues 174-196 of SEQ ID NO:3 or amino acid residues of aC1qC corresponding to amino acid residues 174-196(HTANLCVLLYRSGVKVVTFCGHT) of SEQ ID NO:3; g) amino acid residues 184-192of SEQ ID NO:3 or amino acid residues of a C1qC corresponding to aminoacid residues 184-192 (RSGVKVVTF) of SEQ ID NO:3; h) amino acid residues185-187 of SEQ ID NO:3 or amino acid residues of a C1qC corresponding toamino acid residues 185-187 (SGV) of SEQ ID NO:3; and i) amino acidresidue Ser 185 of SEQ ID NO:3 or an amino acid residue of a C1qCcorresponding to amino acid residue Ser 185 of SEQ ID NO:3.
 8. Themethod of claim 3, wherein the anti-C1q antibody binds specifically toboth human C1q and mouse C1q.
 9. The method of claim 3, wherein theanti-C1q antibody has dissociation constant (K_(D)) for human C1q andmouse C1q of less than 100 pM.
 10. The method of claim 3, wherein theanti-C1q antibody specifically binds to and inhibits a biologicalactivity of C1q.
 11. The method claim 10, wherein the biologicalactivity is (1) C1q binding to an autoantibody, (2) C1q binding to C1r,(3) C1q binding to C1s, (4) C1q binding to phosphatidylserine, (5) C1qbinding to pentraxin-3, (6) C1q binding to C-reactive protein (CRP), (7)C1q binding to globular C1q receptor (gC1qR), (8) C1q binding tocomplement receptor 1 (CR1), (9) C1q binding to beta-amyloid, (10) C1qbinding to calreticulin, (11) activation of the classical complementactivation pathway, (12) activation of antibody and complement dependentcytotoxicity, (13) CH50 hemolysis, (14) synapse loss, (15) B-cellantibody production, (16) dendritic cell maturation, (17) T-cellproliferation, (18) cytokine production (19) microglia activation, (20)Arthus reaction, (21) phagocytosis of synapses or nerve endings, or (22)activation of complement receptor 3 (CR3/C3) expressing cells. 12.(canceled)
 13. The method of claim 1, wherein the antibody is ananti-C1s antibody.
 14. The method of claim 13, wherein the anti-C1santibody is: a) an anti-C1s antibody comprising a light chain variabledomain and a heavy chain variable domain, wherein the light chainvariable domain comprises the HVR-L1, HVR-L2, and HVR-L3 of themonoclonal antibody 5A1 produced by a hybridoma cell line with ATCCAccession Number PTA-120351, or progeny thereof; b) an anti-C1s antibodycomprising a light chain variable domain and a heavy chain variabledomain, wherein the heavy chain variable domain comprises the HVR-H1,HVR-H2, and HVR-H3 of the monoclonal antibody 5A1 produced by ahybridoma cell line with ATCC Accession Number PTA-120351, or progenythereof; c) an anti-C1s antibody comprising a light chain variabledomain and a heavy chain variable domain, wherein the light chainvariable domain comprises the HVR-L1, HVR-L2, and HVR-L3 and the heavychain variable domain comprises the HVR-H1, HVR-H2, and HVR-H3 of themonoclonal antibody 5A1 produced by a hybridoma cell line with ATCCAccession Number PTA-120351 or progeny thereof; d) an anti-C1s antibodycomprising a light chain variable domain and a heavy chain variabledomain, wherein the light chain variable domain comprises the HVR-L1,HVR-L2, and HVR-L3 of the monoclonal antibody 5C12 produced by ahybridoma cell line with ATCC Accession Number PTA-120352, or progenythereof; e) an anti-C1s antibody comprising a light chain variabledomain and a heavy chain variable domain, wherein the heavy chainvariable domain comprises the HVR-H1, HVR-H2, and HVR-H3 of themonoclonal antibody 5C12 produced by a hybridoma cell line with ATCCAccession Number PTA-120352, or progeny thereof; f) an anti-C1s antibodycomprising a light chain variable domain and a heavy chain variabledomain, wherein the light chain variable domain comprises the HVR-L1,HVR-L2, and HVR-L3 and the heavy chain variable domain comprises theHVR-H1, HVR-H2, and HVR-H3 of the monoclonal antibody 5C12 produced by ahybridoma cell line with ATCC Accession Number PTA-120352, or progenythereof; g) an murine anti-human C1s monoclonal antibody 5A1 produced bya hybridoma cell line with ATCC Accession Number PTA-120351, or progenythereof; h) an murine anti-human C1s monoclonal antibody 5C12 producedby a hybridoma cell line with ATCC Accession Number PTA-120352 orprogeny thereof; i) an anti-C1s antibody which binds essentially thesame C1s epitope as the antibody 5A1 produced by a hybridoma cell linewith ATCC Accession Number PTA-120351; or j) an anti-C1s antibody whichbinds essentially the same C1s epitope as the 5C12 antibody produced bya hybridoma cell line with ATCC Accession Number PTA-120352.
 15. Themethod of claim 14, wherein the anti-C1s antibody specifically binds toand inhibits a biological activity of C1s or the C1s proenzyme.
 16. Themethod of claim 15, wherein the biological activity is (1) C1s bindingto C1q, (2) C1s binding to C1r, (3) C1s binding to C2 or C4, (4) theproteolytic enzyme activity of C1s, (5) the conversion of the C1sproenzyme to an active protease, or (6) cleavage of C4, (7) activationof the classical complement activation pathway, (8) activation ofantibody and complement dependent cytotoxicity, or (9) C1F hemolysis.17-20. (canceled)
 21. The method of claim 1, wherein the antibody is ananti-C1r antibody.
 22. The method of claim 1, wherein the antibody is ananti-C1 complex antibody.
 23. The method of claim 1, wherein theantibody binds human C1q, C1r, or C1s, or wherein the antibody bindshuman C1 complex.
 24. (canceled)
 25. The method of claim 1, wherein theantibody is a monoclonal antibody.
 26. The method of claim 1, whereinthe antibody is a mouse antibody, a human antibody, a humanizedantibody, or a chimeric antibody.
 27. The method claim 1, wherein theantibody is an antibody fragment selected from Fab, Fab′-SH, Fv, scFv,and (Fab′)₂ fragments.
 28. The method of claim 1, wherein the antibodyis a bispecific antibody that recognizes a first antigen and a secondantigen.
 29. The method of claim 28, wherein the first antigen isselected from C1q, C1r, C1s, and the C1 complex, and the second antigenis an antigen that facilitates transport across the blood-brain-barrier.30. The method of claim 28, wherein the second antigen is selected fromtransferrin receptor (TR), insulin receptor (HIR), insulin-like growthfactor receptor (IGFR), low-density lipoprotein receptor relatedproteins 1 and 2 (LPR-1 and 2), diphtheria toxin receptor, CRM197, allama single domain antibody, TMEM 30(A), a protein transduction domain,TAT, Syn-B, penetratin, a poly-arginine peptide, an angiopep peptide, orANG1005.
 31. The method of claim 1, wherein the antibody inhibits C3cdeposition, membrane attack complex (MAC) deposition, axonal damageformation, or respiratory muscle damage. 32-34. (canceled)
 35. Themethod of claim 1, wherein the antibody inhibits the classical oralternative complement activation pathway by an amount from at least 30%to at least 99.9%.
 36. (canceled)
 37. The method claim 1, wherein theantibody inhibits complement-dependent cell-mediated cytotoxicity (CDCC)activation pathway by an amount from at least 30% to at least 99.9%. 38.(canceled)
 39. The method of claim 1, wherein the antibody has adissociation constant (K_(D)) for its corresponding antigen from 100 nMto 0.005 nM or less than 0.005 nM.
 40. The method of claim 1, whereinthe antibody inhibits autoantibody-dependent and complement-dependentcytotoxicity (CDC).
 41. The method of claim 1, wherein the antibodyinhibits amplification of the alternative complement activation pathwayinitiated by C1q binding. 42-43. (canceled)
 44. The method of claim 1,further comprising administering a second antibody, wherein the secondantibody is selected from an anti-C1q antibody, an anti-C1r antibody, ananti-C1s antibody and an anti-C1 complex antibody. 45-48. (canceled)